JP2019537430A - Affinity-oligonucleotide conjugates and uses thereof - Google Patents

Abstract

Provided herein are methods and compositions for characterizing a single cell using an affinity-oligonucleotide conjugate. Provided herein are methods and compositions for characterizing a single cell using a tetramer-oligonucleotide conjugate. The methods described herein use sequence readout to analyze proteins in individual cells and are not limited by the number of fluorophores that can be used simultaneously in the same experiment, nor by their spectral overlap.

Description

CROSS REFERENCE This application claims priority under International Patent Application No. PCT / US2016 / 053598, filed Sep. 24, 2016, which is incorporated herein by reference in its entirety. Introduced inside.

background

  Many cell types can be identified and classified by the abundance of a particular set of proteins that are expressed endogenously and located on the cell membrane. This phenomenon allows the study of cells using a process known as immunophenotyping, in which cells are incubated with a fluorescently labeled antibody specific for a known surface protein of the cell, and Binds to various antibodies. Flow cytometry is commonly used to measure the level of surface-bound antibody for each cell. However, flow cytometry based approaches are limited by the number of fluorophores that can be used simultaneously in the same experiment. Furthermore, the number of fluorophores that can be used simultaneously in the same experiment using a flow cytometry-based approach is limited by spectral overlap. Furthermore, flow cytometry does not lend itself to many biologically relevant assays and subsequent DNA sequencing.

Abstract

  Thus, there is a need for a method of characterizing a single cell without these limitations, eg, immunophenotyping. Unlike flow cytometry-based approaches, the methods described herein use sequence readouts to analyze proteins in individual cells, and depending on the number of fluorophores that can be used simultaneously in the same experiment. It is not limited by spectral overlap. In addition, the methods described herein are amenable to many biologically relevant assays and subsequent DNA sequencing. The methods described herein utilize an affinity-oligonucleotide conjugate (eg, an antibody-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate). The oligonucleotide of the conjugate comprises an antigen-ID (AID) sequence barcoded on the surface antigen to which the affinity portion of the affinity-oligonucleotide conjugate specifically binds. Thus, using the methods described herein, single cell antigens (eg, surface proteins) can be analyzed without the need for fluorophores. For example, a single cell surface protein to be displayed can be identified from the antigen ID sequence. As another example, the T cell receptor (TCR) or B cell receptor (BCR) of a single cell presented can be identified from the antigen ID sequence. In some aspects, the binding affinity of a TCR or BCR displayed on the surface of a cell can be determined using an affinity-oligonucleotide conjugate described herein. One or more of the surface proteins of a single cell can be used to define the identity, characteristics or association of a single cell.

  The affinity-oligonucleotide conjugates of the methods described herein provide affinity-oligonucleotides such as slow cell sorting, reduced target yield associated with cell sorting, a limited number of output streams, and affinity. It can be used to overcome the problem of selected bins that do not correspond to the quantified properties of the conjugate. The exemplary affinity-oligonucleotide conjugates shown may replace or enhance sorting using single cell measurements of tetramer binding in the vessel. The exemplary affinity-oligonucleotide conjugates shown can be used in the methods described herein to simultaneously acquire TCR pair sequence, clonal abundance, and relative tetramer affinity. For example, the affinity of the binding of a TCR to a major histocompatibility complex (MHC) -peptide complex of a tetramer-oligonucleotide conjugate to simultaneously acquire a TCR pair sequence that binds to the tetrameric peptide To determine the TCR with a lower binding affinity compared to the TCR with a higher binding affinity to the MHC-peptide complex of the tetramer-oligonucleotide conjugate, Affinity-oligonucleotide conjugates that are oligonucleotide conjugates can be used in the methods described herein. As another example, to simultaneously obtain a BCR pair sequence that binds to a tetrameric B cell receptor antigen, the affinity of BCR binding to the B cell receptor antigen of the tetramer-oligonucleotide conjugate is determined. To determine and to distinguish BCRs with lower binding affinity compared to BCRs with higher binding affinity of tetramer-oligonucleotide conjugates to B cell receptor antigens, Affinity-oligonucleotide conjugates that are oligonucleotide conjugates can be used in the methods described herein.

  Described herein are methods of characterizing, eg, immunophenotyping, cells in vessels (eg, emulsions) using affinity-oligonucleotide conjugates. In some embodiments, the method is used to identify cell subsets in a manner compatible with emulsion-based single cell analysis. In some embodiments, the method is used to identify immune cells specific for the antigen in a manner compatible with emulsion-based single cell analysis. In some embodiments, prior to cell analysis, the surface protein-specific antibody is conjugated to an oligonucleotide. In some embodiments, the oligonucleotide is designed to contain a sequence motif that is unique to the target specificity of the conjugate antibody. In some embodiments, the oligonucleotide is designed to contain a sequence motif that is unique to the target specificity of the conjugate tetramer. The oligonucleotide can be covalently or non-covalently conjugated to the affinity portion of the affinity-oligonucleotide conjugate (eg, an antibody or tetramer) (eg, biotin-oligonucleotide to streptavidin). -Antibodies).

  The method can include incubating the cells with one or more affinity-oligonucleotide conjugates in a mixture or solution. Cells can be washed to remove unbound affinity-oligonucleotide conjugate. The cells are then encapsulated in a vessel, eg, an emulsion. Cells may be present in the vessel at a density of a single cell per vessel. Thus, the affinity-oligonucleotide conjugate in a vessel, eg, a droplet, is bound to the cell surface, eg, via a specific antibody-surface protein interaction. The method can include attaching a vessel-specific DNA sequence (eg, a unique vessel barcode) to the affinity-conjugated oligonucleotide. Further cellular DNA or mRNA analysis, phenotyping, functional testing, cell sorting or other reactions can be performed before, simultaneously with or after barcoding the affinity-conjugated oligonucleotide (eg, with a DNA barcode). Can be implemented.

  The method may include incubating the cells with one or more tetramer-oligonucleotide conjugates in a mixture or solution. Cells can be washed to remove unbound tetramer-oligonucleotide conjugate. The cells are then encapsulated in a vessel, eg, an emulsion. Cells may be present in the vessel at a density of a single cell per vessel. Thus, a tetramer-oligonucleotide conjugate in a vessel, e.g., a droplet, e.g., via a specific TCR-MHC-peptide complex interaction, or a specific BCR-B cell receptor of a tetramer interaction It is bound to the cell surface via body antigens. The method can include attaching a vessel-specific DNA sequence (eg, a unique vessel barcode) to the tetramer-conjugated oligonucleotide. Prior to, simultaneously with or after further cellular DNA or mRNA analysis, phenotyping, functional testing, cell sorting or other reactions barcode the tetramer-conjugated oligonucleotide (eg, with a DNA barcode). Can be implemented.

  The method can include, for example, extracting the nucleic acid from the emulsion after an emulsion experiment. Extracted nucleic acids can be prepared for sequencing and sequenced (eg, using next generation sequencing techniques). The method can include the step of sequencing a polynucleotide molecule from the vessel that contains both the antigen ID sequence and the droplet-specific barcode sequence. The antigen ID sequence can define a specific cell surface protein bound by the oligonucleotide conjugate antibody. The antigen ID sequence can define a specific antibody of the oligonucleotide-conjugated antibody that binds to a particular cell surface protein. The antigen ID sequence can define a specific MHC-peptide complex of the oligonucleotide conjugate tetramer that binds to a particular cellular TCR. The antigen ID sequence can define a specific B cell receptor antigen of the oligonucleotide conjugate tetramer that binds to a particular cellular BCR. Thus, the antigen ID sequence can indicate which surface proteins on the analyzed cells are expressed. In vessels with a single cell, all sequences containing the shared droplet-specific barcode sequence are associated with a single cell. Thus, a single cell can be analyzed to present a set of surface proteins that can be used to define its identity, characteristics or relevance. For example, a single cell can be analyzed to display a TCR with a certain affinity for the MHC-peptide complex, which can also identify the sequence of the TCR, or a single cell can Can be analyzed to present a BCR with a certain affinity for the tetrameric B cell receptor antigen, which can also identify the sequence of the BCR.

  In one aspect, a method is provided that includes performing a reaction in a plurality of vessels, wherein the reaction comprises transforming a Bessel bar-coded polynucleotide comprising a Bessel barcode sequence into a vessel of one of the plurality of vessels. And attaching to the oligonucleotide of the affinity-oligonucleotide conjugate bound to the target antigen of the isolated single cell. In one aspect, a method is provided that includes performing a reaction in a plurality of vessels, wherein the reaction comprises transforming a Bessel bar-coded polynucleotide comprising a Bessel barcode sequence into a vessel of one of the plurality of vessels. Attaching the isolated tetramer-oligonucleotide conjugate oligonucleotide to the TCR of a single cell. In one aspect, a method is provided that includes performing a reaction in a plurality of vessels, wherein the reaction comprises transforming a Bessel bar-coded polynucleotide comprising a Bessel barcode sequence into a vessel of one of the plurality of vessels. Attaching the isolated tetramer-oligonucleotide conjugate oligonucleotide to the BCR of a single cell.

  In one aspect, provided herein is a method comprising performing a reaction in one of a plurality of vessels, wherein the reaction comprises binding a Bessel bar-coded polynucleotide comprising a Bessel bar-coded sequence to an affinity. The affinity-oligonucleotide conjugate binds to a target antigen expressed by a cell in the vessel of the plurality of vessels. In one aspect, provided herein is a method comprising performing a reaction in one of a plurality of vessels, wherein the reaction comprises converting a Bessel bar-coded polynucleotide comprising a Bessel barcode sequence to a four-vessel. Attaching to the oligonucleotide portion of the multimer-oligonucleotide conjugate, wherein the tetramer-oligonucleotide conjugate binds to a TCR expressed by a cell in the vessel of the plurality of vessels. In one aspect, provided herein is a method comprising performing a reaction in one of a plurality of vessels, wherein the reaction comprises converting a Bessel bar-coded polynucleotide comprising a Bessel barcode sequence to a four-vessel. Attaching to the oligonucleotide portion of the multimer-oligonucleotide conjugate, wherein the tetramer-oligonucleotide conjugate binds to the BCR expressed by cells in the vessel of the plurality of vessels.

  In some embodiments, the cells are single cells contained within a vessel. In some embodiments, the vessel comprises two or more vessels of the plurality of vessels. In some embodiments, the vessel comprises each vessel of the plurality of vessels. In some embodiments, the reaction occurs in two or more of the plurality of vessels. In some embodiments, the cells in each vessel are from the same sample. In some embodiments, the cells in one of the first plurality of vessels of the two or more vessels are the cells of the two or more vessels. Derived from the same sample as cells in one of the second plurality of vessels. In some embodiments, the oligonucleotide portion includes an antigen identification sequence (AID). In some embodiments, the AID is barcoded on the target antigen, or on the affinity portion of the affinity-oligonucleotide conjugate. In some embodiments, the AID is barcoded to a TCR with known specificity, or an MHC-peptide complex of a tetramer-oligonucleotide conjugate. In some embodiments, the AID is barcoded on a BCR with known specificity, or on a B cell receptor antigen of a tetramer-oligonucleotide conjugate.

  In some embodiments, the oligonucleotide further comprises a target antigen, or an antigen identification sequence (AID) barcoded on the affinity portion of the affinity-oligonucleotide conjugate. In some embodiments, the oligonucleotide further comprises a TCR with known specificity, or an antigen identification sequence (AID) barcoded on the MHC-peptide complex of the tetramer-oligonucleotide conjugate. In some embodiments, the antigen identification sequence (AID) is a known sequence.

  In some embodiments, the vessel barcoding polynucleotide is from a template vessel barcoding polynucleotide in the vessel.

  In some embodiments, the method further comprises sequencing the oligonucleotide or its amplicon to obtain sequence information.

  In some embodiments, the method further comprises determining a characteristic of the single cell based on the sequence information. In some embodiments, the sequence information includes an antigen identification (AID) sequence. In some embodiments, the method further comprises determining a characteristic of the single cell based on the sequence information. In some embodiments, the feature is a phenotype. In some embodiments, the phenotype is an immunophenotype. In some embodiments, the feature is affinity. In some embodiments, the affinity is an MHC-peptide complex. In some embodiments, the affinity is the relative affinity of the MHC-peptide complex (eg, a TCR with a higher binding affinity compared to a TCR with a lower binding affinity). In some embodiments, the affinity is of a BCR. In some embodiments, the affinity is the relative affinity of the BCR (eg, a BCR with a higher binding affinity compared to a BCR with a lower binding affinity).

  In some embodiments, the method further comprises contacting the affinity-oligonucleotide conjugate with a plurality of cells, including a single cell. In some embodiments, the method further comprises contacting the tetramer-oligonucleotide conjugate with a plurality of cells, including a single cell. In some embodiments, the step of contacting is before a single cell in the vessel is isolated. In some embodiments, the method further comprises washing the plurality of cells after the contacting.

  In some embodiments, the vessel does not include an affinity-oligonucleotide conjugate that is not bound to a target antigen. In some embodiments, the vessel does not include a tetramer-oligonucleotide conjugate that is not attached to a TCR. In some embodiments, the vessel does not include a tetramer-oligonucleotide conjugate that is not attached to a BCR.

  In some embodiments, the method further comprises isolating the single cell in the vessel. In some embodiments, a single cell is bound to the affinity-oligonucleotide conjugate prior to isolation.

  In some embodiments, the method further comprises lysing the single cell. In some embodiments, lysing is after a single cell is isolated in the vessel.

  In some embodiments, the plurality of cells are a plurality of unsorted cells. In some embodiments, the plurality of cells are a plurality of sorted cells. In some embodiments, the plurality of cells is a plurality of enriched cells.

  In some embodiments, the Bessel barcoding polynucleotide of the first vessel of the plurality of vessels or the vessel barcode sequence of the amplicon thereof is the vessel barcode sequence of the second vessel of the plurality of vessels. It differs from the Bessel barcode sequence of the encoding polynucleotide or its amplicon. In some embodiments, each Bessel barcoding polynucleotide in a single one of the plurality of vessels or the amplicon's Bessel barcode sequence comprises the same Bessel barcode sequence. In some embodiments, each Bessel barcoding polynucleotide in any single vessel of the plurality of vessels and the vessel barcode sequence of the amplicon thereof is included in any other unit of the plurality of vessels. It is unique for each vessel barcoding polynucleotide in one vessel and the vessel barcode sequence of its amplicon.

  In some embodiments, the method further comprises attaching the Bessel bar-encoding polynucleotide to a cell polynucleotide from a single cell. In some embodiments, attaching the vessel bar-encoding polynucleotide to the affinity-oligonucleotide conjugate oligonucleotide and attaching the vessel bar-encoding polynucleotide to a cell polynucleotide from a single cell. Doing is performed simultaneously.

  In some embodiments, the method further comprises amplifying the oligonucleotide or its complement. In some embodiments, the method further comprises amplifying the cellular polynucleotide or its complement. In some embodiments, amplifying the oligonucleotide or its complement and amplifying the cellular polynucleotide or its complement are performed simultaneously.

  In some embodiments, the Bessel barcode sequence of the cellular polynucleotide and the oligonucleotide is the same.

  In some embodiments, the method further comprises pooling the oligonucleotide or its amplicon from two or more vessels of the plurality of vessels. In some embodiments, the method further comprises pooling the oligonucleotide or its amplicon and the cellular polynucleotide or its amplicon from two or more vessels of the plurality of vessels. In some embodiments, pooling is prior to sequencing.

  In some embodiments, the affinity-oligonucleotide conjugate comprises a plurality of different affinity-oligonucleotide conjugates. In some embodiments, each of the plurality of affinity-oligonucleotide conjugates comprises a unique antigen identification (AID) sequence. In some embodiments, the oligonucleotide is an affinity molecule barcode (AMB) sequence that is barcoded into a single affinity-oligonucleotide conjugate molecule of the plurality of affinity-oligonucleotide conjugate molecules. including. In some embodiments, each affinity-oligonucleotide conjugate molecule of the plurality of affinity-oligonucleotide conjugate molecules comprises a unique affinity molecule barcode (AMB) sequence.

  In some embodiments, the methods and conjugates described herein comprise a plurality of different tetramer-oligonucleotide conjugates. In some embodiments, each tetramer-oligonucleotide conjugate of the plurality of affinity-oligonucleotide conjugates comprises a unique antigen identification (AID) sequence. In some embodiments, the oligonucleotide is an affinity molecule barcoded barcoded on a single tetramer-oligonucleotide conjugate molecule of one of the plurality of tetramer-oligonucleotide conjugate molecules. (AMB) sequence. In some embodiments, each of the plurality of tetramer-oligonucleotide conjugate molecules comprises a unique affinity molecule barcode (AMB) sequence.

  In some embodiments, the oligonucleotide comprises a fusion sequence, and attaching comprises attaching a Bessel bar-encoding polynucleotide to the fusion sequence. In some embodiments, the oligonucleotide comprises a primer binding sequence. In some embodiments, the oligonucleotide comprises a constant sequence.

  In some embodiments, the method further comprises sequencing the oligonucleotide, its complement, its amplified product, or a combination thereof, thereby producing an oligonucleotide sequence read. Including. In some embodiments, the method further comprises comparing the one or more first oligonucleotide sequence reads to the one or more second oligonucleotide sequence reads. In some embodiments, the method further comprises analyzing the oligonucleotide sequence reads. In some embodiments, the method further comprises analyzing the Bessel barcode sequence of the oligonucleotide sequence read. In some embodiments, the method further comprises analyzing the antigen identification (AID) sequence of the oligonucleotide sequence read. In some embodiments, the method further comprises analyzing the affinity molecule barcode (AMB) sequence of the oligonucleotide sequence read. In some embodiments, analyzing comprises determining the frequency of one or more Bessel barcode sequences, one or more AID sequences, one or more affinity molecule barcode (AMB) sequences, or a combination thereof. Including doing. In some embodiments, analyzing comprises comparing. In some embodiments, the method further comprises comparing the antigen identification (AID) sequence of the oligonucleotide sequence read with the affinity molecular barcode (AMB) sequence of the oligonucleotide sequence read.

  In some embodiments, the method comprises sequencing the cellular polynucleotide, its complement, its amplified product, or a combination thereof, thereby producing a cellular polynucleotide sequence read. Further included. In some embodiments, the method further comprises comparing the oligonucleotide sequence read to a cellular polynucleotide sequence read. In some embodiments, the method further comprises comparing the Bessel barcode sequence of the oligonucleotide sequence read to the Bessel barcode sequence of the cellular polynucleotide sequence read. In some embodiments, the method further comprises comparing the cellular polynucleotide sequence reads. In some embodiments, the method further comprises analyzing the Bessel barcode sequence of the cellular polynucleotide sequence reading. In some embodiments, the method further comprises analyzing the molecular barcode sequence of the cellular polynucleotide sequence read.

  In some embodiments, the method further comprises determining a characteristic of the cell based on the analyzing or comparing. In some embodiments, the method further comprises selecting an antibody, BCR or TCR based on the oligonucleotide sequence reading. In some embodiments, the method comprises selecting an antibody, BCR or TCR based on reading the polynucleotide sequence of the cell.

  In some embodiments, the method determines the affinity of the tetramer for the TCR or BCR and / or the affinity of the TCR or BCR for the tetramer based on the analyzing or comparing steps. The method further includes a step. In some embodiments, the number of sequencing reads indicates the affinity of the tetramer for TCR or BCR. In some embodiments, a higher number of sequence reads for one single cell is greater than a single cell having a lower number of sequence reads for the tetramer-oligonucleotide conjugate and And / or exhibit a higher binding affinity of TCR or BCR. A tetramer binding affinity can indicate binding of a tetrameric MHC-peptide complex or a tetrameric B cell receptor antigen.

  In some embodiments, the method of determining the affinity of a TCR or BCR for a tetrameric oligonucleotide conjugate comprises the step of determining one or more TCRs in the presence of the one or more tetramer oligonucleotide conjugates. Incubating the cells, wherein each of the plurality of tetrameric oligonucleotide conjugates can include a unique identifier barcode. In some embodiments, the method comprises the step of isolating each cell-tetramer complex and the oligonucleotide conjugated to the tetramer complex described herein or a complement thereof. Sequencing at least a portion of the sequence. In some embodiments, the method further comprises counting the number of sequencing reads obtained during the step of sequencing each unique barcode sequence. In some embodiments, the method further comprises analyzing the sequencing information or comparing to a standard curve. In some embodiments, the method comprises: sequencing the affinity of the TCR or BCR for the tetramer, or the affinity of the tetramer for the TCR or BCR, or analyzing or comparing the sequencing information. And determining based on the step of performing the determination. In some embodiments, the Vessel barcoding polynucleotide attached to the oligonucleotide and the Vessel barcoding polynucleotide attached to the cellular polynucleotide are derived from the same template Vessel barcoding polynucleotide in the vessel. . In some embodiments, the Vessel barcoding polynucleotide attached to the oligonucleotide is an amplification product of the template Vessel barcoding polynucleotide.

  In some embodiments, the Vessel barcoding polynucleotide attached to the cellular polynucleotide is an amplification product of the template Vessel barcoding polynucleotide.

  In some embodiments, the vessel includes a solid support. In some embodiments, the vessel does not include a solid support. In some embodiments, each vessel of the plurality of vessels contains a single cell. In some embodiments, the vessel is a well, emulsion or droplet. In some embodiments, the template vessel bar-encoding polynucleotide is not attached to a solid support. In some embodiments, the template vessel bar-encoding polynucleotide is bound to a solid support.

  In some embodiments, the method further comprises attaching a molecular barcoding sequence of one of the plurality of molecular barcoding polynucleotides to the cellular polynucleotide. The coding sequence is barcoded into a single cellular polynucleotide molecule and its amplicon.

  In some embodiments, attaching comprises ligating the Bessel polynucleotide to the oligonucleotide. In some embodiments, attaching comprises attaching the Bessel polynucleotide to the oligonucleotide using an enzyme. In some embodiments, attaching comprises hybridizing the Bessel polynucleotide to the oligonucleotide. In some embodiments, attaching further comprises extending the oligonucleotide. In some embodiments, attaching comprises amplifying the template Vessel bar-encoding polynucleotide.

  In some embodiments, the oligonucleotide is double-stranded. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide is DNA. In some embodiments, the oligonucleotide is an RNA.

  In some embodiments, the cellular polynucleotide comprises a variable region sequence. In some embodiments, the method further comprises pairing the native strand sequence comprising the variable region sequence. In some embodiments, the cellular polynucleotide is DNA. In some embodiments, the cellular polynucleotide is RNA. In some embodiments, the RNA is an mRNA.

  In some embodiments, the single cell is a B cell. In some embodiments, the single cell is a T cell.

  In some embodiments, the affinity portion of the affinity oligonucleotide conjugate binds to a single cell extracellular antigen. In some embodiments, the single cell extracellular antigen is an antigen specific for immune cells. In some embodiments, the single cell extracellular antigen is a T cell specific antigen. In some embodiments, the extracellular antigen is CD4. In some embodiments, the extracellular antigen is CD8. In some embodiments, the single cell extracellular antigen is a B cell specific antigen. In some embodiments, the extracellular antigen is an immunoglobulin.

  In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is an antibody or a fragment thereof. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is a peptide. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is a protein. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is an aptamer. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is a small molecule. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is a drug. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate is a cell. In some embodiments, the cells are antigen presenting cells (APC). In some embodiments, the affinity portion of the affinity oligonucleotide conjugate comprises an MHC-peptide complex. In some embodiments, the affinity portion of the affinity oligonucleotide conjugate comprises a major histocompatibility complex (MHC). In some embodiments, the MHC is in soluble and / or multimeric (eg, tetrameric) form. In some embodiments, the MHC is linked to a peptide. In some embodiments, the peptide is a synthetic peptide. In some embodiments, the MHC binds to a T cell receptor (TCR) and / or a TCR-like binding molecule, eg, a TCR-like antibody or immunoglobulin or chimeric antigen receptor, for example, in a single cell.

  In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate comprises an MHC-peptide complex. In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate comprises major histocompatibility complex (MHC) in soluble and / or multimeric (eg, tetrameric) form. In some embodiments, the MHC is bound to a streptavidin protein, which complexes with a central biotin protein, allowing up to four MHC-streptavidin complexes to be linked to a multimeric complex. (Eg, as shown in FIG. 10A). In some embodiments, the MHC is associated with a peptide. In some embodiments, the MHC is associated with the peptide via a covalent or non-covalent linkage. In some embodiments, the peptide is a synthetic peptide. In some embodiments, the peptide is a synthetic peptide. In some embodiments, the MHC binds to a TCR and / or TCR-like binding molecule, eg, a TCR-like antibody or immunoglobulin or chimeric antigen receptor, eg, in a single cell. In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate comprises the B cell receptor antigen in soluble and / or multimeric (eg, tetrameric) form. In some embodiments, the B cell receptor antigen has been previously administered to the cell or subject. In some embodiments, the B cell receptor antigen binds to a BCR and / or BCR-like binding molecule, eg, a chimeric antigen receptor, eg, of a single cell.

  In some embodiments, the affinity moiety is an antigen-recognition molecule and / or an immunoreceptor, such as an antibody or immunoglobulin or a portion or fusion thereof, an engineered immunoreceptor, a chimeric antigen receptor (CAR) Or specifically binds to the TCR. In some such embodiments, the affinity portion comprises an antibody or receptor, such as an antigen or epitope recognized by CAR, or portions thereof. In some embodiments, the affinity portion comprises an antibody or antigen-binding fragment thereof that specifically binds to an immune receptor. In some aspects, the antibody or antigen-binding fragment thereof specifically binds to a variable and / or antigen-binding portion of a receptor, eg, an idiotope. In some aspects, the affinity molecule is an anti-idiotype antibody or a fragment thereof.

  In some embodiments, the affinity portion of the affinity oligonucleotide conjugate comprises a major histocompatibility complex (MHC) or a functional or binding portion thereof. In some embodiments, the affinity moiety comprises a multimer of the MHC, optionally a tetramer of the MHC. In some embodiments, the MHC is in a soluble form. In some embodiments, the MHC is bound to and / or comprises a peptide within the groove of the MHC. In some embodiments, the peptide is a synthetic peptide. In some embodiments, the MHC binds to a single cell T cell receptor (TCR). In some embodiments, the affinity moiety comprises an antibody or a peptide that binds to a chimeric antigen receptor (CAR), and / or the target is an antibody or a CAR. In some embodiments, the affinity moiety is or comprises an antigen or epitope specifically recognized by the antibody or chimeric antigen receptor and / or an antibody that specifically binds to it, optionally And anti-idiotype antibodies that specifically bind to its antigen binding portion.

  In one aspect, a single cell from a sample comprising a plurality of cells, an affinity-oligonucleotide conjugate bound to a target antigen of a single cell, and a Vessel barcoding polynucleotide comprising a Vessel barcode sequence are each There is provided a composition comprising a plurality of vessels comprising: In some embodiments, the Bessel bar-encoding polynucleotide or its complement is attached to an oligonucleotide of an affinity-oligonucleotide conjugate.

  In one aspect, a single cell from a sample comprising a plurality of cells, a tetramer-oligonucleotide conjugate linked to the TCR or BCR of a single cell, and a Vessel bar-coded poly- Compositions are provided that include a plurality of vessels, each containing a nucleotide. In some embodiments, the Vessel bar-encoding polynucleotide or its complement is attached to an oligonucleotide of a tetramer-oligonucleotide conjugate. In one aspect, a composition comprising a single lysed cell from a sample comprising a plurality of cells and a plurality of vessels each comprising an affinity-oligonucleotide conjugate bound to a target antigen of the single lysed cell is provided. Provided; the oligonucleotides of the affinity-oligonucleotide conjugate comprise a Bessel barcode sequence, and cellular polynucleotides from a single lysed cell comprise the same Bessel barcode sequence.

  In one aspect, a composition comprising a single lysed cell from a sample comprising a plurality of cells, and a plurality of vessels each comprising a tetramer-oligonucleotide conjugate bound to a TCR or BCR of the single lysed cell. The oligonucleotide of the tetramer-oligonucleotide conjugate comprises a Bessel barcode sequence, and cellular polynucleotides from a single lysed cell comprise the same Bessel barcode sequence. In one aspect, provided herein is a composition comprising a plurality of vessels, wherein one of the vessels comprises a single cell from a sample comprising a plurality of cells, and a vessel barcode sequence. The vessel further comprises an affinity-oligonucleotide conjugate, or an oligonucleotide portion derived therefrom, that binds to a single cell target antigen.

  In one aspect, provided herein is a composition comprising a plurality of vessels, wherein one vessel of the plurality of vessels comprises a single cell from a sample comprising a plurality of cells, and a vessel bar comprising a vessel barcode sequence. The encoding polynucleotide, wherein the vessel further comprises a tetramer-oligonucleotide conjugate or oligonucleotide portion derived therefrom that binds to the TCR or BCR of a single cell.

  In some embodiments, the reaction occurs in two or more of the plurality of vessels. In some embodiments, the vessel comprises each vessel of the plurality of vessels. In some embodiments, the plurality of vessels comprises two or more vessels. In some embodiments, the cells in each vessel are from the same sample. In some embodiments, the cells in one of the first plurality of vessels of the two or more vessels are the cells of the two or more vessels. Derived from the same sample as cells in one of the second plurality of vessels. In some embodiments, the Bessel bar-encoding polynucleotide or its complement is attached to an oligonucleotide of an affinity-oligonucleotide conjugate. In some embodiments, the Vessel bar-encoding polynucleotide or its complement is attached to an oligonucleotide of a tetramer-oligonucleotide conjugate. In some embodiments, a single cell is lysed.

  In one aspect, provided herein is a composition comprising a plurality of vessels, wherein one of the plurality of vessels comprises a single lysed cell from a sample comprising a plurality of cells, and a single lysed cell from a sample comprising a plurality of cells. An affinity-oligonucleotide conjugate comprising an affinity portion that binds to a target antigen of the invention, or comprising an oligonucleotide portion of the affinity-oligonucleotide conjugate; the oligonucleotide portion of the affinity-oligonucleotide conjugate comprises a vessel bar. The coding polynucleotide sequence, a cellular polynucleotide from a single lysed cell contains the same vessel bar coding sequence.

  In one aspect, provided herein is a composition comprising a plurality of vessels, wherein one vessel of the plurality of vessels comprises a single lysed cell from a sample comprising a plurality of cells, and a single lysed cell. A tetramer-oligonucleotide conjugate comprising an affinity portion that binds to a TCR or BCR of a cell, or comprising the oligonucleotide portion of the tetramer-oligonucleotide conjugate; an oligonucleotide of a tetramer-oligonucleotide conjugate Portions include a Bessel barcode sequence and cellular polynucleotides from a single lysed cell include the same Bessel barcode sequence.

  In one aspect, a first container comprising a first oligonucleotide comprising a first antigen identification (AID) sequence, wherein the first AID sequence is a known sequence; a second container; A second container comprising a second oligonucleotide comprising an antigen identification (AID) sequence, wherein the second AID sequence is a known sequence and different from the first AID sequence; One or more comprising a reagent capable of conjugating one oligonucleotide to a first affinity molecule and a reagent capable of conjugating a second oligonucleotide to a second affinity molecule. A plurality of third containers; how to conjugate the first oligonucleotide to the first affinity molecule, and how to conjugate the second oligonucleotide to the second affinity molecule. Describe A kit comprising a set of instructions is provided.

  In one aspect, a first container comprising a first oligonucleotide comprising a first antigen identification (AID) sequence, wherein the first AID sequence is a known sequence; a second container; A second container comprising a second oligonucleotide comprising an antigen identification (AID) sequence, wherein the second AID sequence is a known sequence and different from the first AID sequence; One or more reagents comprising a reagent capable of conjugating one oligonucleotide to a first tetramer and a reagent capable of conjugating a second oligonucleotide to a second tetramer. A third container; a kit comprising a set of instructions describing a method of conjugating the first oligonucleotide to the first tetramer and the second oligonucleotide to the second tetramer molecule. Provided.

  In one aspect, a first container comprising an oligonucleotide comprising an antigen identification (AID) sequence, wherein the AID sequence is a known sequence; wherein the oligonucleotide is conjugated to an affinity molecule. A second container containing possible reagents; a third container containing a plurality of vessel bar-encoding polynucleotides; and one of the plurality of vessel bar-encoding polynucleotides when conjugated to the affinity molecule. Kits are provided that include a set of instructions that describe how to attach the Bessel bar-coded polynucleotide to the oligonucleotide.

  In one aspect, a first container comprising an oligonucleotide comprising an antigen identification (AID) sequence, wherein the AID sequence is a known sequence; wherein the oligonucleotide is conjugated to a tetramer. A second container containing the possible reagents; a third container containing the plurality of vessel bar-encoding polynucleotides; and one of the plurality of vessel bar-encoding polynucleotides when conjugated to the tetramer. Kits are provided that include a set of instructions that describe how to attach a vessel bar-coded polynucleotide to an oligonucleotide.

  The methods and compositions disclosed herein can be used for tumor profiling. For example, these methods can include linking the cellular phenotype to an immune repertoire in a patient sample to identify tumor-reactive TCRs. The methods and compositions disclosed herein can be used for adoptive cell therapy. For example, these methods can include genetic analysis of T cells without sorting. For example, these methods can include combining T cell cloning information (using the TCR) with gene expression patterns during product manufacture and processing. In some embodiments, the methods disclosed herein track, characterize, monitor, and monitor adoptively transferred cells obtained from a patient before, during, or after adoptive cell therapy. And / or used to evaluate. The methods and compositions disclosed herein can be used to identify a TCR against a known target. For example, these methods can include identifying high affinity clones that may respond highly to the antigen but do not grow well. The methods and compositions disclosed herein can be used for multiplexing cell samples. For example, an emulsion comprising a pooled cell sample that has been contacted with one or more affinity-oligonucleotide conjugates can be used to identify the original cell sample while simultaneously processing multiple samples.

  The methods and compositions disclosed herein can be used for measuring multiplexed TCR or BCR affinity by sequencing. The methods and compositions disclosed herein can be used to quantify the binding affinity of a single cell TCR or BCR to the tetramer of a tetramer-oligonucleotide conjugate. Further, the quantification may be sensitive to tetramer-oligonucleotide conjugate concentration, wherein the binding affinity signal may be dependent on tetramer-oligonucleotide conjugate concentration (eg, , A decrease in tetramer-oligonucleotide conjugate concentration results in a corresponding decrease in binding affinity signal). Further, the methods and compositions disclosed herein can be used to distinguish antigen-specific cells from non-specific cells. For example, distinguish between cells having a TCR or BCR that binds to a tetramer of a tetramer-oligonucleotide conjugate and cells having a TCR or BCR that does not bind to a tetramer of a tetramer-oligonucleotide conjugate. thing. Further, one advantage of the methods and compositions disclosed herein is that non-specific cells (ie, tetramers) can be used without the use of flow cytometry gating to separate those two populations. -The ability to distinguish a population of antigen-specific cells (i.e., tetramers + cells) from cells. Often, flow cytometry gating may be the "best guess" of population separation based on the separation of tetramer-population from tetramer-population on a flow cytometry plot, but that is not the case. It may be accurate, and thus may result in loss of cells from each population or mischaracterization of cells. In contrast, the methods and compositions described herein do not rely on gating techniques to distinguish populations of cells, but instead individually quantify the binding affinity of each cell in a sample It may be a high throughput method. In addition, the methods and compositions disclosed herein provide for cells having a TCR with high binding affinity to MHC-peptides (i.e., high binding agents) and TCRs with low binding affinity to MHC-peptides To distinguish between high and low binding affinity cells in a sample, as well as to distinguish cells having low binding affinity (ie, low binding agents), as well as using the high throughput methods described herein. Can be used to

  Embedding by reference

  All publications, patents, and patent applications referred to in this specification are for all purposes, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The entirety of which are incorporated herein by reference.

  The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of features described herein may be found in the following detailed description, which illustrates illustrative examples in which the principles of the features described herein are utilized. Can be obtained by referring to the drawings.

FIG. 1 shows an exemplary schematic of a vessel of the method described herein.

FIG. 2 shows an exemplary design of an oligonucleotide tag conjugated to an antibody. Each colored block represents a portion of the complete oligonucleotide sequence. The fusion sequence is used for enzymatic attachment of a droplet-specific DNA barcode within the emulsion reaction. Although only one possible arrangement of the sequence is shown, other arrangements are compatible with the methods described.

FIG. 3A shows an exemplary simultaneous capture of immunoreceptor sequences with additional mRNA and protein targets. Surface protein targets are quantified by preincubating cells with DNA-labeled stained antibodies prior to emulsion sequencing.

FIG. 3B shows exemplary CD4 and CD8 mRNA and protein measurements of 3,682 droplet barcode TCR VαVβ pairs generated from healthy human T cells.

FIG. 3C shows an exemplary match between mRNA and protein measurements (each point is a droplet barcode linked to a TCR VαVβ pair).

FIG. 3D shows an exemplary table of the simultaneous detection of CD4 and CD8 mRNA and protein from unsorted T cells in an emulsion. From 30,000 input T cells, 3,682 TCR pairs were recovered. The matrix shows the frequency of TCR pairs that were considered CD4 ⁺ or CD8 ⁺ by mRNA versus protein (based on molecular counting, majority rule).

FIG. 3E shows exemplary results of 45,870 single cell TCR pairs using an affinity-oligonucleotide conjugate targeting CD4 and an affinity-oligonucleotide conjugate targeting CD8.

FIG. 4 shows a schematic of an exemplary method in which an affinity-oligonucleotide conjugate targeting CD4 and an affinity-oligonucleotide conjugate targeting CD8 are used.

FIG. 5 illustrates the results of the single immune cell barcoding method in an emulsion. FIG. 5A is an illustration of passing two aqueous streams containing a cell and a lysis / reaction (LR) mixture through an oil to produce a monodisperse emulsion in excess of 8 million droplets per hour.

FIG. 5B is an exemplary diagram showing that cells in the vessel are lysed and subjected to molecule-specific and droplet-specific barcoding in a single reaction.

FIG. 5C is an exemplary diagram showing that target mRNA is reverse transcribed and template switch tagged with a universal adapter sequence. Thereafter, PCR amplification of the droplet barcode template, initially diluted to about one molecule per droplet, occurs. The amplified barcode is added to the template-switched cDNA by a complementary overlap extension method. The product is recovered from the emulsion and purified using the RT primer biotin prior to additional library processing steps and high-throughput sequencing.

FIG. 5D shows that double barcoding allows clustering of sequencing reads into their derived molecules and derived droplets, while minimizing sequencing errors and amplification bias. FIG. 4 is an exemplary diagram showing that pairings are reconstructed.

FIG. 6 illustrates the results of a method for recovering BCR from isolated healthy B cells. FIG. 6A is an illustration of an example of a droplet in which 3 million B cells passed through the emulsion at 0.2 cells / droplet, resulting in approximately 90% of occupied cells containing single cells. .

6B _is a diagram of an exemplary V H _{V L} pairing accuracy. After the emulsion barcode grant and sequencing data, enriched for data derived from a single cell droplets, the V _H V _L pairing accuracy was assessed using a pair consistency expanding clones.

FIG. 6C is an exemplary graph of percent droplet barcode versus Ig isotype. 259,368 pieces of (most abundant isotype of each within the droplet) heavy chain isotype of the filtered V _H V _L pairs and light chain loci utilization is shown.

FIG. 6D is an exemplary graph of the rank abundance of the 100 most frequent heavy chain clones in each of the six independent emulsion fractions. The overall frequency of 0.05% is marked.

FIG. 6E is an exemplary graph of _VH vs. _VL expression in cells as assessed by the number of captured mRNAs in each droplet barcode. For each isotype, 5,000 points are shown.

FIG. 6F is an exemplary graph of _VH vs. _VL mutation correlation of BCR pairs and density distribution within each isotype.

FIG. 7 illustrates the results of a method for discovering HIV broadly neutralizing antibodies (bNAbs). Figure 7A is an illustration of a graph of the heavy chain isotype distribution of 38,620 pieces of recovered V _H V _L pair derived from the HIV long-term non-progressors B cells encased in the emulsion. A small percentage of IgG chains align well with previously known bNAbs ("PGT-like").

FIG. 7B is an illustration of an exemplary phylogenetic tree of complete VDJ amino acid sequences of known bNAbs (black) + freshly recovered (red, labeled with a droplet barcode). Heavy (left) and light (right) chains are plotted separately. Potentially mismatched antibodies PGT122 and PGT123 are blue.

FIG. 7C is an illustration of the neutralizing activity (IC ₅₀ , μg / mL) of eight newly discovered PGT-like variants against 10 strains of HIV compared to a control stock of PGT121.

FIG. 8 illustrates the results of a method for characterizing TILs from ovarian tumors. FIG. 8A is an illustration of an example of a droplet in which 400,000 unsorted, isolated cells from an ovarian tumor were placed in an emulsion and BCR and TCR pairs were simultaneously collected by emulsion barcode application. It is.

FIG. 8B is an exemplary graph of a droplet barcode versus receptor chain combination showing the number of all V _H / V _L and Vα / Vβ combinations observed in the droplet barcode after filtering.

FIG. 8C is an exemplary graph of percent droplet barcode versus heavy chain isotype distribution of the recovered BCR pairs.

FIG. 8D is an exemplary graph of _VH vs. _VL mutational correlations for BCR pairs and density distribution within each isotype.

FIG. 8E shows an exemplary graph of the total (top) TCR and BCR pairs and the number of captured mRNAs of different isotypes (bottom).

FIG. 8F is an exemplary graph of clone analysis showing the rank abundance of the 30 most frequent BCR heavy chain clones (top) and the 30 most frequent TCR beta chain clones in each of the six independent emulsion fractions. Is shown. Overall frequency levels of 1% and 10% are shown.

FIG. 9 illustrates a method for immunophenotyping using an antibody-oligonucleotide conjugate. FIG. 9A shows an exemplary schematic showing two vessels, each containing a single cell, attached to an antibody-oligonucleotide conjugate. (DB1-droplet barcode 1; DB2-droplet barcode 2; MB1-molecular barcode 1; MB2-molecular barcode 2; AID-antigen ID barcode; AMB1-antibody molecule barcode 1; AMB2-antibody molecule Barcode 2).

FIG. 9B shows an exemplary schematic showing two vessels, each containing an RNA molecule from the lysed cells of the vessel of FIG. 9A. The RNA molecule is reverse transcribed and a non-template nucleotide is added to the end of the cDNA molecule created by the reverse transcription. A molecular barcode is hybridized to a non-template nucleotide added to the end of the cDNA molecule created by reverse transcription.

FIG. 9C shows an exemplary schematic depicting two vessels, each containing a template barcoded polynucleotide, that have been amplified and attached to the cDNA of the vessel of FIG. 9B by hybridization. The cDNA has been extended (top). Thereafter, the extended cDNA is amplified (lower).

FIG. 9D shows an exemplary schematic showing that RNA-MB-DB species with the same molecular barcode (MB) attached to the same RNA sequence are likely to be the result of PCR replication. RNA-MB-DB species with two different MBs attached to the same identical RNA sequence (RNA1-MB1-DB and RNA1-MB2-DB) are the two original independent RNA molecules and are derived from PCR replication Absent.

FIG. 9E shows that a DB-AMB-AID species with the same antibody molecule barcode (AMB) attached to a sequence with the same droplet barcode (DB) and antigen ID barcode (AID), FIG. 9 shows an exemplary schematic diagram showing that the result is likely to be high. The DB1-AMB1-AID1 and DB1-AMB2-AID1 species with two different AMBs attached to an array with the same droplet barcode (DB) and antigen ID barcode (AID) have the same unit in the vessel. Two independent oligonucleotide molecules, each derived from two independent antibody oligonucleotide conjugate molecules, each having an antibody that specifically binds to the same target antigen attached to one cell and not from PCR replication. Two different AIDs, DB1-AMBn-AID1 and DB1-AMBn-AID2, attached to a sequence with the same droplet barcode (DB) and the same or different antibody molecule barcode (AMB) are placed in the vessel. Two independent antibody oligonucleotide conjugate molecules attached to the same single cell of the antibody, wherein one of the antibody oligonucleotide conjugate molecules specifically binds to the first target antigen The other antibody oligonucleotide conjugate molecule having an antibody that binds has an antibody that specifically binds to a second target antigen.

FIG. 10A shows a schematic diagram of an exemplary affinity-oligonucleotide conjugate of the method described herein. As indicated, exemplary affinity-oligonucleotide conjugates can include an MHC-peptide affinity moiety. In some aspects, such a conjugate can be referred to as a tetramer-oligonucleotide conjugate.

FIG. 10B shows a schematic of an exemplary affinity-oligonucleotide conjugate of the method described herein. In some aspects, such a conjugate can be referred to as a tetramer-oligonucleotide conjugate.

FIG. 11A shows an exemplary graph of the binding signal of two exemplary affinity-oligonucleotide conjugates of the methods described herein, including an affinity moiety that binds a TCR.

FIG. 12A shows an exemplary schematic diagram of a T cell bound to an exemplary affinity-oligonucleotide conjugate of the methods described herein.

FIG. 12B shows an exemplary schematic of T cells in a droplet attached to an exemplary affinity-oligonucleotide conjugate of the methods described herein. The nucleic acids in the droplets are marked with a droplet identification sequence and incorporated into a next generation sequencing library.

FIG. 13 shows an exemplary schematic of an oligonucleotide tag conjugated to an exemplary affinity-oligonucleotide conjugate tetramer. A tetramer ID is a short, constant DNA sequence that corresponds to a tetramer batch and allows for the multiplexing of different targets, such as peptide-MHC targets, in a single experiment. Molecular barcodes are degenerate sequences that allow molecular counting to quantify bound tetramers.

FIG. 14 shows a schematic of an exemplary affinity-oligonucleotide conjugate generated from a DNA-labeled MHC tetramer reagent (tetramer-oligonucleotide conjugate). In one embodiment, a Cy5-linked DNA oligonucleotide is synthesized and conjugated to streptavidin or neutravidin. In one embodiment, a non-fluorescent DNA oligonucleotide is conjugated to APC-streptavidin. In one embodiment, a mixture of a non-fluorescent DNA oligonucleotide and streptavidin or neutravidin is conjugated to activated APC.

FIG. 15 shows an exemplary method for conjugating an oligonucleotide to the affinity portion of an affinity-oligonucleotide conjugate using click chemistry.

FIG. 16 shows a schematic of an exemplary workflow for preparing and characterizing exemplary affinity-oligonucleotides.

FIG. 17 shows an example method described herein using six different affinity-oligonucleotide conjugates targeting CD3, CD19, CD4, CD8, HLA-DR, and CTLA-4. The results are shown. Each point is a droplet barcode / single cell. Cell identity was revealed by the type of receptor pair recovered (TCR = T cells; Ig = B cells).

FIG. 18 shows a further image of an exemplary tetramer-oligonucleotide conjugate. In some embodiments, the tetramer-oligonucleotide comprises a Fluorophore. In some embodiments, the tetramer-oligonucleotide does not contain a fluorophore.

FIG. 19 shows exemplary binding curves of two exemplary tetramer-oligonucleotide conjugates of the methods described herein containing an MHC-peptide affinity moiety that binds to a TCR.

FIG. 20 shows that target-specific primary or non-target-specific primary CD8 + T cells were incubated with either standard pMHC tetramer reagents or DNA-labeled pMHC tetramers (tetramer-oligonucleotide conjugates). 3 shows a flow cytometry plot. DNA-labeled pMHC tetramers were used at decreasing concentrations, as indicated by the triangle immediately above the three panel columns on the right.

FIG. 21 shows non-binding TCRs (eg, TCRs not specifically recognized by tetramer-oligonucleotide conjugate reagents) and binding TCRs (eg, TCRs specifically recognized by tetramer-oligonucleotide conjugate reagents). 4 shows a comparison of the number of tetramer-oligonucleotide conjugates per cell between cells displaying different TCRs. As shown along the X-axis, increasing concentrations of tetramer-oligonucleotide conjugate were used.

Detailed description

  Some aspects are described below, by way of illustration, in connection with an example application. It should be understood that numerous specific details, relevance and methods have been set forth in order to provide a thorough understanding of the features described herein. However, one of ordinary skill in the art readily recognizes that the features described herein may be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts may occur in a different order and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.

  The terminology used herein is for the purpose of describing particular cases only, and is not intended to be limiting. As used herein, the singular forms “a”, “an”, and “the” also include the plural unless the context clearly dictates otherwise. Is intended. Further, the terms "including", "includes", "having", "has", "with", or variants thereof, are intended to refer to the detailed description and / or To the extent used in any of the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

  The term "about" or "approximately" refers to how the value is measured or determined, i.e., within an allowable error range for a particular value determined by one of ordinary skill in the art, depending in part on the limitations of the measurement system. It can mean that For example, "about" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within a factor of five or less, more preferably within a factor of two or less. Where a particular value is recited in the present application and claims, the term "about", unless otherwise indicated, should be assumed to be within the permissible error range for that particular value. .

  Provided are methods and compositions for phenotyping a single cell (eg, an immune cell) (eg, in an emulsion) using an affinity-oligonucleotide conjugate (eg, an antibody-oligonucleotide conjugate). Is the object of the present invention.

Definition

  Thus, the term "antibody" herein is used in the broadest sense and includes fragment antigen binding (Fab) fragment, F (ab ') 2 fragment, Fab' fragment, Fv fragment, recombinant IgG. Intact antibodies and functional (antigen-binding) antibodies thereof, including (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibody (eg, sdAbs, sdFv, Nanobodies) fragments Polyclonal and monoclonal antibodies, including fragments, are included. This term refers to genetically engineered and / or otherwise modified forms of immunoglobulins, eg, intrabodies, peptibodies, chimeric, fully human, humanized, and heteroconjugate antibodies, multispecific Gender, including bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD.

  The terms “complementarity determining region” and “CDR”, synonymous with “hypervariable region” or “HVR,” refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Is known in the art. Generally, three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3) Exists. It is known in the art that "framework regions" and "FR" refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-H-FR) in each full-length light chain variable region. L2, FR-L3 and FR-L4).

  The exact amino acid sequence boundaries of a given CDR or FR can be found in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" number). Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262: 732-745 (1996). , "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell. receptor variable domains and Ig superfamily V-like domains ", Dev Comp Immunol, January 2003; 27 (1): 55-77 (" I GT "numbering scheme), and Honegger A and Pluckthun A," Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool ", J Mol Biol, June 8, 2001; 309 (3): 657-70, ("Aho" numbering scheme), and can be readily determined using any of several well-known schemes.

  The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with the insertion provided by the insertion letter, such as "30a" and deletion, appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on an analysis of the crystal structure of the complex and is similar in many respects to the Chothia numbering scheme.

  Table A below shows exemplary positions of the boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and Contact schemes, respectively. Are listed. For CDR-H1, residue numbering is listed using both Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, FR-L1 is located between CDR-L1 and CDR-L2, and so on. The end of the Chothia CDR-H1 loop when numbered using the indicated Kabat numbering convention depends on the length of the loop, since the indicated Kabat numbering scheme places the insertion at H35A and H35B. Note that it fluctuates between H32 and H34.

  Thus, unless stated otherwise, a given antibody or region thereof, eg, the “CDR” or “complementarity determining region” of its variable region, or each specified CDR (eg, CDR-H1, CDR-H2) Should be understood to encompass certain (or particular) complementarity determining regions, as defined by any of the above schemes. For example, if a particular CDR (e.g., CDR-H3) is stated to include the amino acid sequence of the corresponding CDR in a given VH or VL amino acid sequence, then such CDR is defined by any of the above schemes. The corresponding CDRs within the variable region (e.g., CDR-H3). In some embodiments, the identified CDR sequences are identified.

  Similarly, unless otherwise specified, the FRs of a given antibody or region thereof, eg, the variable region thereof, or the individual identified FR (s) (eg, FR-H1, FR-H2) are known in the art. Should be understood to encompass certain (or specific) framework regions defined by any of the following. In some examples, a particular CDR, FR, or scheme for the identification of FR (s) or CDR (s) is identified, such as CDRs defined by the method of Kabat, Chothia or Contact. In other cases, a specific amino acid sequence of a CDR or FR is provided.

  The terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs ( See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

  Among the provided antibodies, mention may be made of antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments are formed from Fv, Fab, Fab ', Fab'-SH, F (ab') 2; diabodies; linear antibodies; single-chain antibody molecules (eg, scFv); and antibody fragments. Includes but is not limited to multispecific antibodies. In certain embodiments, these antibodies are single chain antibody fragments that include a variable heavy and / or variable light chain region, eg, a scFv.

  Unless otherwise specified, the term "TCR" should be understood to encompass the entire TCR as well as antigen-binding portions or fragments thereof (also referred to as MHC-peptide binding fragments). In some embodiments, the TCR is an intact or full-length TCR. In some embodiments, the TCR is less than the full-length TCR, but binds to a specific antigenic peptide, ie, in the context of an MHC molecule, ie, an MHC-peptide complex, that binds to an MHC molecule. It is an antigen-binding portion. In some cases, the antigen-binding portion or fragment of a TCR may include only a portion of the full-length or intact TCR structural domain, but still bind an intact TCR (eg, an MHC-peptide complex). It is possible to combine with In some cases, the antigen-binding portion or fragment of the TCR comprises a variable domain of the TCR sufficient to form a binding site for binding to a specific MHC-peptide complex, eg, a variable α of the TCR. Chain and a variable β chain, for example, where, in general, each chain contains three complementarity determining regions. Included are polypeptides or proteins having a binding domain that is or is homologous to an antigen binding domain. Complementarity determining region (CDR) -grafted antibodies and TCRs and other humanized antibodies and TCRs, including CDR and framework region modifications, are also contemplated by these terms. Although reference may be made only to immunoglobulin chains (eg, heavy and light chains), the disclosed invention is directed to a plurality of other different types of paired sequences, eg, T cell receptor chain pairs (TCRα It should be noted that the present invention can be applied to immunoglobulins and TCRβ chains and TCRγ chains and TCRδ chains).

  The ability of T cells to recognize antigens associated with various cancers or infectious organisms is composed of both alpha (α) and beta (β) chains or both gamma (γ) and delta (δ) chains. Is given by the TCR. The proteins that make up these chains are encoded by DNA that uses a unique mechanism to generate the greatest diversity of TCRs. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to peptides presented by MHC class I and II proteins on the surface of antigen presenting cells (APCs). Binding of the TCR to an antigenic peptide on the APC is a central event in T cell activation, which occurs at the immune synapse at the point of contact between the T cell and the APC.

  Each TCR contains a variable complementarity determining region (CDR) as well as a framework region (FR). The amino acid sequences of the third complementarity determining region (CDR3) loop of the α and β chain variable domains are the variable (Vβ) gene segment, the diversity (Dβ) gene segment, and the joining (Jβ) gene at the β chain locus, respectively. The sequence diversity of αβ T cells resulting mainly from recombination between segments, as well as between similar Vα and Jα gene segments at the α chain locus, is determined. The presence of multiple such gene segments at the α and β chain loci of the TCR allows a large number of distinct CDR3 sequences to be encoded. Independent addition and deletion of nucleotides at the Vβ-Dβ, Dβ-Jβ and Vα-Jα junctions during the process of TCR gene rearrangement further increases CDR3 sequence diversity. In this regard, immunocompetence is reflected in the diversity of the TCR.

Immunoglobulins expressed by B cells (Ig) is, in some _embodiments, consists of four polypeptide chains forming a H 2 _{L 2} structure, two heavy chains (IgH) and two light chains (IgL) It is a protein. Each pair of IgH and IgL chains contains a hypervariable domain consisting of the _VL and _VH regions, and a constant domain. Ig heavy chains of Ig are of several types, μ, δ, γ, α and β. Ig diversity within an individual is determined primarily by the hypervariable domains. Like the TCR, the V domain of the IgH chain is created by the combinatorial ligation of _VH , _DH and _JH gene segments. _V H _{-D H,} independent additions and deletions of nucleotides in the D H -J _H and _V H -J _H joints during the process of Ig gene rearrangement further increases the diversity of the hypervariable domain sequence . Here, immunocompetence is reflected in Ig diversity.

  The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs ( See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using the VH or VL domains from the antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

  "Affinity moiety" refers to that portion of an affinity-oligonucleotide conjugate that interacts with a target antigen. Exemplary affinity moieties include antibodies, peptides, proteins, aptamers, small molecules, drugs, cells, MHC, and the like.

  "Hypervariable region" refers to the amino acid residues of an antibody or TCR that are responsible for antigen binding. This hypervariable region contains complementarity determining regions or amino acid residues from the CDRs. Framework or FR residues are those variable domain residues other than the hypervariable region residues as defined herein.

  Among the antibodies provided, antibody fragments are among others. An “antibody fragment” refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments are formed from Fv, Fab, Fab ', Fab'-SH, F (ab') 2; diabodies; linear antibodies; single-chain antibody molecules (eg, scFv); and antibody fragments. Includes but is not limited to multispecific antibodies. In certain embodiments, the antibody is a single chain antibody fragment comprising a variable heavy and / or light chain region, eg, a scFv.

  Single domain antibodies are antibody fragments that contain all or part of the antibody heavy chain variable domain, or all or part of the antibody light chain variable domain. In certain embodiments, a single domain antibody is a human single domain antibody.

  Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of intact antibodies, as well as production by recombinant host cells. In some embodiments, these antibodies are recombinantly produced fragments, eg, fragments containing non-naturally occurring configurations, eg, two or more antibodies linked by a synthetic linker, eg, a peptide linker. Fragments having regions or chains and / or fragments that may not be produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, these antibody fragments are scFvs.

  Also provided are TCR fragments, including antigen-binding fragments. In some embodiments, the TCR is a variant of a full-length TCR that does not include its transmembrane and / or cytoplasmic region (s), which may be referred to as an antigen-binding portion thereof, eg, a completely soluble TCR. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (scTCR), for example, a scTCR having a structure as described in PCT Patent Publication Nos. WO 03/020563, WO 04/033685 or WO 2011/044186. In certain embodiments, the TCR is a single chain TCR fragment that includes an alpha chain variable region linked to a beta chain variable region, eg, scTv. In some embodiments, the scTv is also called a scFv.

A single-chain Fv or scFv is, in some aspects, a variable heavy (V _H ) and variable light (V _L ) domain of an antibody, or a variable alpha or gamma chain (Vα or Vγ) and a variable beta or TCR of a TCR. Refers to antibodies or TCR fragments that contain a delta chain (Vβ or Vδ) domain, wherein these domains are present in a single polypeptide chain. Generally, Fv polypeptide between a polypeptide linker which enables to form the desired structure for antigen binding sFv is between V _H and V _L domains, or the Vα domain and Vβ domains, Alternatively, it further comprises between the Vγ domain and the Vδ domain.

Diabodies, in some aspects, refer to small antibodies and / or TCR fragments that have two antigen-binding sites, which fragments are connected to a _VL in the same polypeptide chain ( _VH - _VL ). _VH , or Vα connected to Vβ in the same polypeptide chain (Vα-Vβ), or Vγ connected to Vδ in the same polypeptide chain (Vγ-Vδ). By using a linker that is too short to allow pairing between two domains on the same chain, those domains pair with complementary domains on another chain and create two antigen-binding sites Forced to do so. Exemplary diabodies are more fully described, for example, in EP 404097 and WO 93111161.

  Bispecific antibodies or bispecific TCRs, in some aspects, refer to antibodies or TCRs that exhibit specificity for two different types of antigen. These terms as used herein specifically include, but are not limited to, antibodies and TCRs that exhibit binding specificity for a target antigen and for another target that facilitates delivery to a particular tissue. Not done. Similarly, multispecific antibodies and TCRs have two or more binding specificities.

The straight-chain antibody or a linear TC, in some embodiments, tandem Fd segments which form a pair of antigen binding regions _{(e.g., V H -C H1 -V H -C} H1 or Vα-Cα ₁ -Vα- Cα ₁ ). Linear antibodies and TCRs can be bispecific or monospecific, for example, as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995).

An antigen binding domain, in some aspects, refers to one or more fragments of an antibody or TCR that retains the ability to specifically bind an antigen. Non-limiting examples of antibody fragments included within such terms include (i) a Fab fragment which is a monovalent fragment consisting of the V _L , V _H , C _L and C _Hl domains; (ii) a disulfide bridge in the hinge region. A single arm of the antibody comprising: a F (ab ') ₂ fragment, which is a bivalent fragment comprising two Fab fragments joined by: (iii) a Fd fragment consisting of the _VH and _CH1 domains; (iv) a scFv included and (vi) an isolated CDR;: Fv fragment containing the _{V L} and _{V H} domains, (v) dAb fragments containing _{V H} domain (544 546 pages Ward et al, (1989) Nature 341 volumes) But not limited to these. In addition, antibodies comprising a single heavy chain and a single light chain, or TCRs having a single alpha or single beta chain are included in this definition.

“F (ab ′) ₂ ” and “Fab ′” moieties can be produced by treating Ig with proteases such as pepsin and papain, which reside between the hinge regions in each of the two heavy chains. Antibody fragments produced by digesting immunoglobulins in the vicinity of the disulfide bond. For example, papain cleaves IgG upstream of a disulfide bond present between the hinge regions in each of the two heavy chains to produce a light chain composed of V _L and C _L and V _H and C _Hγ1 (heavy chain The heavy chain fragment, which is composed of the γ1 region in the constant region of, produces two homologous antibody fragments that are connected via a disulfide bond in their C-terminal region. Each of these two homologous antibody fragments is called a 'Fab'. Pepsin also cleaves IgG downstream of the disulfide bond present between the hinge regions in each of the two heavy chains, slightly larger than the fragment where the two aforementioned 'Fabs' are connected in the hinge region Generate antibody fragments. This antibody fragment is called F ('ab') ₂ . The Fab fragment also contains the first constant domain of the constant domain and the heavy chain of light chain (C H _1). 'Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain C _{H 1} domain including one or more cysteine (s) from the antibody hinge region differ from Fab fragments. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains bear a free thiol group. F (ab ') ₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them.

Fv refers, in some aspects, to an antibody or TCR fragment that contains a complete antigen recognition and binding site. This region is from a tightly non-covalently associated dimer of one heavy and one light chain variable domain or one TCRα and one TCRβ or one TCRγ and one TCRδ chain. Become. In this configuration, the three CDR of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer or V.alpha-V? Dimers or V.gamma-V8 dimers. Collectively, one or more combinations of CDRs from each of the _VH and _VL chains or the Vα-Vβ or Vγ-Vδ chains confer antigen binding specificity to the antibody or TCR. For example, CDRH3 and CDRL3 are expressed when the antibody or TCR is transferred to the _VH and _VL chains or Vα and Vβ or Vγ-Vδ chains of the recipient's selected antibody, TCR or antigen-binding fragment thereof. It is understood that this combination of CDRs can be tested for binding, affinity, etc. Even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) is capable of recognizing and binding to an antigen, but more than when combined with a second variable domain. Are also likely to have low affinity. In addition, the two domains of the Fv fragment ( _VL and _VH or Vα and Vβ or Vγ and Vδ) are encoded by separate genes, which are _VL and _VH or Vα and Vβ or Vγ and Vδ. A single protein chain in which the chain regions combine to form a monovalent molecule (known as a single-chain Fv (scFv); Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc Natl. Acad. Sci. USA 85: 5879-5883; and Osbourn et al. (1998) Nat. Biotechnol. 16: 778) with synthetic linkers to allow them to be made. Can be linked using the method. Such scFvs are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any _VH and _VL sequences of a specific scFv can be ligated to the cDNA or genomic sequence of the Fc region to generate an expression vector that encodes a complete Ig (eg, IgG) molecule or other isotype. _VH and _VL can also be used in the production of Fab, Fv or other fragments of Ig using either protein chemistry or recombinant DNA technology.

Antigen binding polypeptides can also include heavy chain dimers, for example, antibodies from camelids and sharks. Camelid and shark antibodies contain a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain). Since the _VH region of heavy chain dimeric IgG in camelids does not interact hydrophobically with the light chain, the region in the heavy chain that normally contacts the light chain is changed to a hydrophilic amino acid residue in camelids. Is done. The _VH domain of a heavy chain dimer IgG is called a _VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (called V-NAR domain) and five C-like constant domains (C-NAR domain). The camelid, diversity of antibody repertoire is determined by CDR1,2 and 3 in _{V H} or _{V HH} region. CDR3 in the camel V _HH region is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7 (9): 1129).

  A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. A `` humanized form '' of a non-human antibody is a humanized form of a non-human antibody that has been humanized to typically reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody Refers to the variant. In some embodiments, some FR residues in a humanized antibody are non-human (e.g., antibodies from which CDR residues are derived, e.g., to restore or improve antibody specificity or affinity). ) Is replaced with the corresponding residue from

  Among the antibodies provided, human antibodies are particularly preferred. A “human antibody” is an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a non-human source utilizing sequences encoding humans or human cells, or other human antibodies, such as a human antibody repertoire or human antibody library. Is an antibody having The term excludes humanized forms of non-human antibodies that include a non-human antigen binding region, eg, those in which all or substantially all CDRs are non-human.

  Human antibodies can be prepared by administering an immunogen to an intact human antibody, or a transgenic animal that has been modified to produce an intact antibody having human variable regions, in response to an antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci that replace the endogenous immunoglobulin loci or that are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display libraries and cell-free libraries, containing antibody coding sequences from the human repertoire.

  Among the antibodies provided, monoclonal antibodies, including monoclonal antibody fragments, may be mentioned. The term "monoclonal antibody" as used herein refers to an antibody derived from or within a substantially homogeneous population of antibodies, i.e., the individual antibodies that comprise the population are naturally occurring. Except for possible variants that contain the mutations present or occur during the production of the monoclonal antibody preparation, such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. This term should not be interpreted as requiring production of the antibody by any particular method. Monoclonal antibodies can be made by a variety of techniques, including but not limited to production from hybridomas, recombinant DNA methods, phage display and other antibody display methods.

  The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Provided antibodies and antibody chains and polypeptides, including other peptides, eg, linkers and binding peptides, can include amino acid residues, including natural and / or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may include alterations to the native or native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutation of the host producing the protein or errors due to PCR amplification.

A "germline sequence" refers to a gene sequence from the germline (haploid gametes and the diploid cells from which they are formed). Germline DNA comprises multiple gene segments that encode a single Ig heavy or light chain, or a single TCRα or TCRβ chain, or a single TCRγ or TCRδ chain. These gene segments are retained in germ cells but cannot be transcribed and translated until they are placed into a functional gene. During the differentiation of B cells and T cells in the bone marrow, these gene segments are randomly shuffled by 10 dynamic genetic system capable of involving a large specificity than ^8. Most of these gene segments are published and collected by germline databases.

Affinity refers to the equilibrium constant for the reversible binding of the two agents, expressed as K _D. The affinity of a binding protein for a ligand, eg, an antibody for an epitope, or, eg, for a TCR for an MCH-peptide complex, can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, about 100 nM. To about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). The term “avidity” refers to the resistance of a complex of two or more drugs to dissociation after dilution.

An epitope refers, in some aspects, to a portion of an antigen or other macromolecule that is capable of forming a binding interaction with a variable region binding pocket of an antibody or TCR. Such binding interactions may be manifested as intermolecular contacts with one or more amino acid residues of one or more CDRs. Antigen binding may involve, for example, the interaction of CDR3s, CDR3 pairs, or, in some instances, up to all six CDRs of the _VH and _VL chains. An epitope may be a linear peptide sequence (ie, “continuous”) or may be composed of non-contiguous amino acid sequences (ie, “conformation” or “discontinuous”). An antibody or TCR can recognize one or more amino acid sequences; thus, an epitope can define more than one distinct amino acid sequence. In some aspects, the TCR can recognize one or more amino acid sequences or epitopes in the context of MHC. Epitopes recognized by antibodies and TCRs can be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. Binding interactions are manifested as intermolecular contacts with one or more amino acid residues of the CDR.

  In some embodiments, reference to an antibody or TCR having specific binding refers to a situation where one antibody or TCR does not show any significant binding to a molecule other than an antigen, including an antibody or an epitope recognized by the TCR. Point. The term is also applicable, for example, when the antigen-binding domain is specific for a particular epitope carried by some antigen, in which case the selected antibody, TCR, or antigen-binding Those antigen-binding fragments that carry the domain can bind to various antigens that carry the epitope. The term “preferentially binds” or “specifically binds” refers to an antibody, TCR, or fragment thereof that binds to an epitope with a higher affinity than if it binds to an unrelated amino acid sequence. If it is cross-reactive with other polypeptides, it means that they are not toxic at the level formulated for administration to human use. In one aspect, such affinity is at least 1-fold, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater than the affinity of the antibody, TCR, or fragment thereof for an unrelated amino acid sequence. Large, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater At least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1000 times greater. The term “bond” refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions under physiological conditions, Include interactions, such as salt and water bridges, and any other conventional means of attachment.

  The term “bond” refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions under physiological conditions, Include interactions, such as salt and water bridges, and any other conventional means of attachment.

  In some aspects, the term “tetramer” can refer to a complex that includes four subunits that are bound to a single molecule of streptavidin, which can bind to a population of cells , Thus identifying it. The subunit may be an MHC-peptide complex. A subunit may be an MHC without an associated peptide. The subunit may be a B cell receptor antigen. The population of cells identified by the tetramer may be a population that expresses a receptor, such as a TCR or BCR, that binds to a subunit of the tetramer. The population of cells may be antigen-specific T cells. The population of cells may be antigen-specific B cells. The tetramer may be fluorescently labeled. As used herein, an MHC-peptide tetramer can be used interchangeably with pMHC.

  "Pharmaceutically acceptable" is physiologically acceptable and typically does not produce an allergic or similar adverse reaction when administered to a human, such as abnormal gastric hyperactivity, dizziness, etc. , Molecular entities and compositions.

  "Prevention" refers to prevention (prophylaxis), prevention of the onset of symptoms, prevention of the development of a disease or disorder associated with excessive levels of protein or correlated with protein activity.

  “Inhibition”, “treatment” and “treating” are used interchangeably and are associated with, for example, stagnation of symptoms, prolongation of survival, partial or complete remission of symptoms, and excessive levels of protein. Or partial or complete eradication of a condition, disease or disorder that correlates with protein activity. For example, treatment of cancer includes, but is not limited to, cancerous growth or stasis of a tumor, partial or complete elimination. Treatment or partial elimination includes, for example, a predetermined factor reduction in growth or tumor size and / or volume, eg, about 2 ×, about 3 ×, about 4 ×, about 5 ×, about 10 ×, about 20 ×. , About 50 times, or any magnification between them. Similarly, treatment or partial elimination includes about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, Percent reductions of 50%, 60%, 70%, 80%, 90%, 95% or any percentage reduction therebetween may be included.

  A neutralizing antibody or neutralizing TCR, in some aspects, refers to any antibody or TCR that inhibits replication of a pathogen, such as a virus or bacterium, regardless of the mechanism by which neutralization is achieved.

  An antibody repertoire or TCR repertoire refers to a collection of antibodies, TCRs or fragments thereof. The antibody repertoire can be used, for example, to select particular antibodies or to screen for particular properties, such as binding ability, binding specificity, ability for gastrointestinal transit, stability, affinity, and the like. The term includes combinatorial, eg, antibody phage display libraries, including, without limitation, single chain Fv (scFv) and Fab antibody phage display libraries from any source, including naive, synthetic and semi-synthetic libraries. Specifically include antibody and TCR libraries, including all forms of the libraries.

  “Target nucleic acid molecule”, “target polynucleotide”, “target polynucleotide molecule” refers to any nucleic acid of interest.

  Polymerase chain reaction (PCR) refers to an in vitro amplification reaction of a polynucleotide sequence by simultaneous primer extension of a complementary strand of a double-stranded polynucleotide. The PCR reaction produces a copy of the template polynucleotide flanked by primer binding sites. The result with the two primers is an exponential increase in template polynucleotide copy number of both strands with each cycle, as both strands are replicated with each cycle. The polynucleotide duplex has an end corresponding to the end of the primer used. PCR can include one or more repetitions of denaturing the template polynucleotide, annealing the primer to the primer binding site, and extending the primer with a DNA or RNA polymerase in the presence of the nucleotide. The particular temperature, duration at each step, and the rate of change between steps depends on many factors well known to those skilled in the art (McPherson et al., IRL Press, Oxford (1991 and 1995)). For example, in conventional PCR using Taq DNA polymerase, double-stranded template polynucleotides can be denatured at temperatures> 90 ° C., primers can be annealed at temperatures ranging from 50-75 ° C., and primers can be It can be stretched at a temperature in the range of 72-78 ° C. In some embodiments, the PCR includes reverse transcription PCR (RT-PCR), real-time PCR, nested PCR, quantitative PCR, multiplex PCR, and the like. In some embodiments, the PCR does not include RT-PCR (U.S. Patent Nos. 5,168,038, 5,210,015, 6,174,670, 6,569). , 627 and 5,925,517; Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002)). RT-PCR involves a PCR reaction preceded by a reverse transcription reaction, in which the resulting cDNA is amplified, and nested PCR means that the amplicons of the first PCR reaction using the first set of primers are Two-step PCR, wherein at least one becomes a sample for a second PCR reaction using a second set of primers that binds to an internal position of the amplicon of the first PCR reaction. Multiplex PCR involves a PCR reaction in which multiple polynucleotide sequences are subjected to PCR simultaneously in the same reaction mixture. The PCR reaction volume can be anywhere between 0.2 pL and 1000 μL. Quantitative PCR involves a PCR reaction designed to determine the absolute or relative amount, abundance, or concentration of one or more sequences in a sample. Quantitative measurements can include comparing one or more reference sequences or standards to a polynucleotide sequence of interest (Freeman et al., Biotechniques, 26: 112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17: 9439-9449 (1989); Zimmerman et al., Biotechniques, 21: 268-279 (1996); Diviacco et al., Gene, 122: 3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17: 9439-9446 (1989)).

  “Nucleotide,” “nucleoside,” “nucleotide residue,” and “nucleoside residue,” as used herein, refer to a deoxyribonucleotide or ribonucleotide residue, or to use in an amplification reaction (eg, a PCR reaction). Other similar nucleoside analogs capable of functioning as components of suitable primers can be meant. Such nucleosides and derivatives thereof can be used as components of the primers described herein, unless otherwise specified. Enhance their stability or usefulness in amplification reactions, provided that the chemical modifications do not interfere with their recognition by the polymerase as deoxyguanine, deoxycytosine, deoxythymidine or deoxyadenine, as appropriate. There is nothing in the present application meant to impede the use of nucleoside derivatives or bases that have been chemically modified to do so. In some embodiments, the nucleotide analog may stabilize hybridization. In some embodiments, nucleotide analogs can destabilize hybridization. In some embodiments, nucleotide analogs can enhance the specificity of hybridization. In some embodiments, nucleotide analogs can reduce the specificity of hybridization.

  “Nucleic acid” or grammatical equivalent refers to either a single nucleotide or at least two nucleotides linked together by a covalent bond.

  "Polynucleotide" or grammatical equivalent refers to at least two nucleotides linked together by a covalent bond. Polynucleotides include molecules that contain two or more nucleotides. Polynucleotides include ribonucleotides, deoxyribonucleotides, including purine and pyrimidine bases, or other natural nucleotide bases, chemically or biochemically modified non-natural nucleotide bases, or derivatives of nucleotide bases. Or a polymeric form of nucleotides of any length, either of peptide nucleic acid (PNA). The backbone of the polynucleotide may contain sugar and phosphate groups, or modified or substituted sugar or phosphate groups. Polynucleotides can include modified nucleotides, for example, methylated nucleotides and nucleotide analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can include other molecules, such as another hybridized polynucleotide. A polynucleotide comprises a sequence of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or both. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including without limitation heterochromatin DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, Includes molecular interfering RNA (siRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of certain sequences, isolated RNA of certain sequences, nucleic acid probes and primers. A polynucleotide may be isolated from a natural source, recombinant, or artificially synthesized.

Polynucleotides can include non-standard nucleotides, for example, nucleotide analogs or modified nucleotides. In some embodiments, non-standard nucleotides can stabilize hybridization. In some embodiments, non-standard nucleotides can destabilize hybridization. In some embodiments, non-standard nucleotides can enhance the specificity of hybridization. In some embodiments, non-standard nucleotides can reduce the specificity of hybridization. Examples of non-standard nucleotide modifications include 2'O-Me, 2'O-allyl, 2'O-propargyl, 2'O-alkyl, 2'fluoro, 2'arabino, 2'xylo, 2'fluoroarabino , Phosphorothioate, phosphorodithioate, phosphoramidate, 2 'amino, 5-alkyl substituted pyrimidine, 3' deoxyguanosine, 5-halo substituted pyrimidine, alkyl substituted purine, halo substituted purine, bicyclic nucleotide, 2'MOE, PNA molecule, LNA-molecule, LNA-like molecule, diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetyl Cytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethyl guanine, 2-methyl-adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, ^{N 6} - adenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2 Thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), Wybutoxosine, shoe Uracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, and derivatives thereof.

  "Subject," "individual," "host," or "patient" refers to a living organism, such as a mammal. Examples of subjects and hosts include horses, cows, camels, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice (eg, humanized mice), gerbils, non-human primates (eg, macaques). Non-mammalian vertebrates, eg, birds (eg, chickens or ducks), fish (eg, sharks) or frogs (eg, Xenopus), and non-mammals, including non-mammalian invertebrates , As well as their transgenic species. In certain aspects, a subject refers to a single organism (eg, a human). In certain embodiments, or alternatively, a group of individuals that make up a small cohort with common immune factors and / or diseases for study and / or a cohort of individuals without disease (eg, a negative / normal control). Is provided. The subject from which the sample is obtained may suffer from a disease and / or disorder (eg, one or more allergies, infections, cancer or autoimmune disorders, etc.) and is compared to a non-diseased, negative control subject Can be done.

  "Kit" refers to a delivery system for delivering materials or reagents to perform the methods disclosed herein. In some embodiments, the kit includes reaction reagents (eg, probes, enzymes, etc. in a suitable container) and / or supporting materials (eg, buffers for performing the assay, written instructions, etc.). Storage systems are included that allow their transport or delivery from one location to another. For example, a kit includes one or more enclosures (eg, boxes) containing the appropriate reaction reagents and / or support materials. Such contents may be delivered together or separately to the intended recipient. For example, a first container may contain enzymes for use in an assay, while a second container contains a plurality of primers.

  Polypeptide refers, in some aspects, to a molecule comprising at least two amino acids. In some embodiments, the polypeptide consists of a single peptide. In some embodiments, the polypeptide comprises two or more peptides. For example, the polypeptide may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, It may comprise 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 peptides or amino acids. Examples of polypeptides include, but are not limited to, amino acid chains, proteins, peptides, hormones, polypeptides, saccharides, lipids, glycolipids, phospholipids, antibodies, enzymes, kinases, receptors, transcription factors and ligands .

  A sample, in some aspects, refers to a biological, environmental, medical, subject, or patient sample, or a sample comprising a polynucleotide, such as a target polynucleotide.

Affinity-oligonucleotide conjugates Affinity-oligonucleotide conjugates include an affinity molecule portion (eg, an antibody or an MHC-peptide complex) and an oligonucleotide portion. The antigen-identifying sequence of the oligonucleotide of the affinity-oligonucleotide conjugate can be used to identify one or more antigens with which the affinity-oligonucleotide conjugate specifically interacts. In some embodiments, the oligonucleotide is covalently attached to the affinity portion of the conjugate. In some embodiments, the oligonucleotide is non-covalently attached to the affinity portion of the conjugate. The affinity-oligonucleotide conjugate may be a tetramer-oligonucleotide conjugate. A tetramer-oligonucleotide conjugate can include a tetramer (eg, a B cell receptor antigen tetramer or an MHC-peptide complex tetramer) and an oligonucleotide moiety. The antigen identification sequence of the oligonucleotide of the tetramer-oligonucleotide conjugate can be used to identify one or more antigens with which the tetramer-oligonucleotide conjugate specifically interacts. In some embodiments, the oligonucleotide is covalently attached to the affinity portion or tetramer of the conjugate. In some embodiments, the oligonucleotide is non-covalently attached to the affinity portion or tetramer of the conjugate.

  In some embodiments, the affinity-oligonucleotide conjugate comprises a single affinity moiety. In some embodiments, the affinity-oligonucleotide conjugate is a multivalent affinity-oligonucleotide conjugate. For example, a multivalent affinity-oligonucleotide conjugate may include the antigen binding domains of at least two affinity molecules conjugated to one or more oligonucleotide (s). For example, a multivalent affinity-oligonucleotide conjugate can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 conjugated to one or more oligonucleotides. , 200, 500 or 1,000 affinity molecules of the antigen binding domain. In some embodiments, the affinity-oligonucleotide conjugate comprises a single oligonucleotide. In some embodiments, the affinity-oligonucleotide conjugate comprises two or more oligonucleotides. For example, an affinity-oligonucleotide conjugate can be at least about 2,3,4,5,6,7,8 conjugated to one or more affinity molecules (eg, an antibody or MHC-peptide complex). , 9, 10, 20, 50, 100, 200, 500 or 1,000 oligonucleotides. In some embodiments, the affinity-oligonucleotide conjugate comprises two or more oligonucleotides comprising the same antigen ID (AID) sequence. For example, an affinity-oligonucleotide conjugate can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500 or 1,000 comprising the same AID sequence. An oligonucleotide may be included.

Affinity-Affinity Portion of Oligonucleotide Conjugate The affinity portion (or domain) of the affinity-oligonucleotide conjugate is the region, molecule, domain, portion (portion) of the affinity-oligonucleotide conjugate that binds to the target antigen. ), Fragments or moieties. Thus, the affinity moiety confers the ability to bind or specifically bind to a given target antigen, eg, the extracellular domain of a cell surface protein. In some embodiments, the affinity portion does not substantially interact with an antigen of another affinity-oligonucleotide conjugate comprising a different antigen ID sequence. In some embodiments, the affinity moiety is a molecule that can include a nucleic acid or a molecule to which an oligonucleotide can be attached without substantially eliminating binding of the affinity moiety to a target antigen.

  The affinity portion of the affinity-oligonucleotide conjugate can be a nucleic acid molecule or can be proteinaceous. Affinity moieties include RNA, DNA, RNA-DNA hybrids, small molecules (eg, drugs), aptamers, polypeptides, proteins, antibodies and fragments thereof, TCRs and fragments thereof, viruses, virus particles, cells, and fragments thereof. And combinations thereof (e.g., Fredriksson et al. (2002) Nat Biotech 20: 473-77; Gullberg et al. (2004) PNAS 101: 8420-24). See). For example, the affinity portion may be a single stranded RNA, a double stranded RNA, a single stranded DNA, a double stranded DNA, a DNA or RNA comprising one or more double stranded regions and one or more single stranded regions, It can be an RNA-DNA hybrid, small molecule, aptamer, polypeptide, protein, antibody, antibody fragment, TCR, TCR fragment, MHC, MHC-peptide complex, viral particle, cell, or any combination thereof.

In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate targets a cell. For example, the affinity portion of an affinity-oligonucleotide conjugate can target T cells or B cells. In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate targets a particular cell type or cell subset. For example, the affinity portion of an affinity-oligonucleotide conjugate can target CD4 ^- T cells or CD8 ⁺ T cells. For example, the affinity portion of an affinity-oligonucleotide conjugate can target T cells that contain a TCR that specifically recognizes a particular antigen. For example, the affinity portion of an affinity-oligonucleotide conjugate can target T cells that contain a TCR that specifically recognizes a particular MHC-peptide complex.

  In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate targets the extracellular domain of a cellular target. For example, the affinity portion of an affinity-oligonucleotide conjugate can target a cell receptor, for example, the extracellular domain of a T cell receptor (TCR). For example, the affinity portion of an affinity-oligonucleotide conjugate can target a glycosylated region of the extracellular domain of a cellular receptor. For example, the affinity portion of an affinity-oligonucleotide conjugate can target a ligand binding region of the extracellular domain of a cellular receptor. For example, the affinity portion of an affinity-oligonucleotide conjugate can target a region of the extracellular domain of a receptor of a cell that does not bind ligand.

  In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate comprises an MHC-peptide complex. In some embodiments, the MHC-peptide complex targets a TCR. In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate comprises a B cell receptor antigen. In some embodiments, the B cell receptor antigen targets a BCR.

Protein In some embodiments, the affinity moiety is a polypeptide, protein, or any fragment thereof. In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate is a protein. In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate is a peptide. For example, the affinity portion of an affinity-oligonucleotide conjugate can be an antibody, eg, a binding domain of an antibody. For example, the affinity portion of an affinity-oligonucleotide conjugate can be an MHC-peptide complex. For example, the affinity moiety can be a purified polypeptide, an isolated polypeptide, a fusion-tagged polypeptide, a polypeptide attached to a cell or a membrane of a virus or virion, or a polypeptide spanning them, a cytoplasmic protein, Intracellular protein, extracellular protein, kinase, phosphatase, aromatase, helicase, protease, oxidoreductase, reductase, transferase, hydrolase, lyase, isomerase, glycosylase, extracellular matrix protein, ligase, ion transporter, channel, pore, apoptotic protein , Cell adhesion proteins, pathogenic proteins, abnormally expressed proteins, transcription factors, transcription regulators, translation proteins, chaperones, secreted proteins, Gand, hormone, cytokine, chemokine, nuclear protein, receptor, transmembrane receptor, signaling factor, antibody, membrane protein, integral membrane protein, superficial membrane protein, cell wall protein, globular protein, fibrous protein, sugar It can be a protein, lipoprotein, chromosomal protein, any fragment thereof, or any combination thereof. In some embodiments, the affinity moiety is a heterologous polypeptide. In some embodiments, the affinity moiety is a protein that is overexpressed in cells using molecular techniques such as transfection. In some embodiments, the affinity moiety is a recombinant polypeptide. For example, the affinity moiety can include a sample (eg, a protein that is overexpressed by an organism) produced in bacteria (eg, E. coli), yeast, mammals or insect cells. In some embodiments, the affinity moiety is a polypeptide comprising a mutation, insertion, deletion, or polymorphism. In some embodiments, the affinity moiety is an antigen, eg, a polypeptide used to immunize an organism or generate an immune response in an organism, eg, for the production of antibodies.

  In some embodiments, the affinity portion of the tetramer-oligonucleotide conjugate is a tetramer. In some embodiments, the tetrameric subunit is a protein. In some embodiments, the tetrameric subunit is a peptide. In some embodiments, the tetrameric subunit is a complex of proteins and / or peptides. In some embodiments, the protein and / or peptide complex may be covalently or non-covalently linked and / or associated. For example, the affinity portion of a tetramer-oligonucleotide conjugate may be an MHC-peptide complex. Non-limiting examples of some of the peptides in an MHC-peptide complex include purified polypeptide, isolated polypeptide, fusion-tagged polypeptide, cell or polypeptide attached to the membrane of a virus or virion. Peptides or polypeptides that span them, cytoplasmic proteins, intracellular proteins, extracellular proteins, kinases, phosphatases, aromatase, helicases, proteases, oxidoreductases, reductases, transferases, hydrolases, lyases, isomerases, glycosylases, extracellular matrix proteins, Ligases, ion transporters, channels, pores, apoptotic proteins, cell adhesion proteins, pathogenic proteins, abnormally expressed proteins, transcription factors, transcription regulators, translation proteins Quality, chaperone, secreted protein, ligand, hormone, cytokine, chemokine, nuclear protein, receptor, transmembrane receptor, signal transduction factor, antibody, membrane protein, integral membrane protein, superficial membrane protein, cell wall protein, It can be a peptide derived from a globular protein, fibrous protein, glycoprotein, lipoprotein, chromosomal protein, any fragment thereof, or any combination thereof. In some embodiments, the peptide is derived from a disease associated antigen. For example, in some aspects, the peptide is derived from or derived from a tumor-associated antigen, such as a universal tumor antigen, a virus-associated antigen, an autoimmunity-associated antigen, an inflammation-associated antigen, a bacteria-associated antigen, or any combination thereof. be able to. In some embodiments, the affinity moiety is a protein that is overexpressed in cells using molecular techniques such as transfection. In some embodiments, the affinity moiety is a recombinant polypeptide. For example, an affinity moiety can include a sample (eg, a protein that is overexpressed by an organism) produced in bacteria (eg, E. coli), yeast, mammals or insect cells. In some embodiments, the affinity moiety is a polypeptide containing a mutation, insertion, deletion, or polymorphism. In some embodiments, the affinity moiety is an antigen, eg, a polypeptide used to immunize an organism or generate an immune response in an organism, eg, for the production of antibodies. As another example, the affinity portion of a tetramer-oligonucleotide conjugate may be a B cell receptor antigen. The B cell receptor antigen may be a tetrameric subunit.

Antibodies In some embodiments, the affinity moiety is an antibody, eg, a binding fragment of an antibody. Antibodies can specifically bind to specific spatial and polar mechanisms of another molecule. For example, the antibody can be a purified antibody, an isolated antibody, a fragment of an antibody, or a fusion-tagged antibody. In some embodiments, the antibodies are overexpressed in cells using molecular techniques such as transfection. In some embodiments, the antibodies are recombinant antibodies. Antibodies can specifically bind to certain spatial and polar mechanisms of another molecule, such as a cell surface molecule. Antibodies can be monoclonal, polyclonal or recombinant, and can be secreted by techniques well known in the art, such as immunization of the host and collection of serum (polyclonal), or by preparing continuous hybrid cell lines. It can be prepared by collecting the protein (monoclonal) or by cloning and expressing a nucleotide sequence encoding at least the amino acid sequence required for specific binding of the native antibody or a mutagenized version thereof. In addition, aggregates, polymers, and conjugates of immunoglobulins or fragments thereof can be used as needed, as long as the binding affinity for a particular molecule is maintained. Examples of antibody fragments include Fab fragments, which are monovalent fragments consisting of the _VL , _VH , _CL, and _CH1 domains; F, which is a bivalent fragment, comprising two Fab fragments joined by a disulfide bridge in the hinge region. (Ab) ₂ fragments; Fd fragment consisting of V _H and C _H1 domains; Fv fragment consisting of V _L and V _H domains of a single arm of the antibody; single domain antibody (dAb) fragment consisting of V _H domain (Ward) (1989) Nature 341: 544-46); and isolated CDRs and single-chain fragments (scFv) (single-chain fragments) in which the _VL and _VH regions combine to form a monovalent molecule. Known as chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242: 423-26; and Huston et al., (1988) PNAS 85: 5879-83. Irradiation of it) is included. Thus, antibody fragments include Fab, F (ab) ₂ , scFv, Fv, dAb, and the like. Although the two domains _VL and _VH are encoded by separate genes, they are synthesized using artificial peptide linkers that allow them to be made as a single protein chain, using recombinant methods. Can be linked. Such single chain antibodies include one or more antigen binding portions. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies. The antibody can be human, humanized, chimeric, isolated, dog, cat, donkey, sheep, any plant, animal or mammal.
MHC

In some cases, recognition of antigenic structures by the cellular immune system is mediated by surface expressed major histocompatibility complex (MHC). Cells, such as antigen presenting cells (APCs), may in some aspects be present in the specific peptide binding fold of the MHC molecule, and thus, in some aspects, into short peptides that can be recognized by T cells. Process proteins such as antigens. Specific recognition of epitopes (peptide fragments) by the T cell receptor (TCR) generally requires simultaneous interaction with MHC molecules. Stable multimeric complexes can be prepared using MHC protein subunits containing bound peptides. MHC-antigen complexes can form stable structures with T cells that recognize the complex via their antigen receptor, thereby allowing binding to T cells that specifically recognize the antigen I do. The affinity portion of the affinity-oligonucleotide conjugate, or of the tetramer-oligonucleotide conjugate, can target T cells. The affinity portion of the affinity-oligonucleotide conjugate can specifically target T cells. The affinity portion of the affinity-oligonucleotide conjugate, or of the tetramer-oligonucleotide conjugate, can target a T cell receptor or a TCR-like molecule, such as a TCR-like CAR. The affinity portion of the affinity-oligonucleotide conjugate can specifically target the T cell receptor. For example, the affinity portion of the affinity-oligonucleotide conjugate, or of the tetramer-oligonucleotide conjugate, can include an MHC molecule. For example, the affinity portion of an affinity-oligonucleotide conjugate, or of a tetramer-oligonucleotide conjugate, can include an MHC-peptide complex (MHC-p). For example, the affinity portion of an affinity-oligonucleotide conjugate or of a tetramer-oligonucleotide conjugate can have the formula (ABP) _n , where A is MHC class I or a α chain of MHC class II protein, B is for beta chain or MHC class I protein class II MHC proteins are beta ₂ microglobulin, P is a peptide. In some embodiments, n is 1. In some embodiments, n is greater than or equal to two. The MHC protein subunit may be in a soluble form. For example, a soluble MHC protein subunit can be derived from a native MHC protein subunit by deletion of the transmembrane domain or a portion thereof. In some embodiments, the MHC protein subunit does not include a cytoplasmic domain. In some embodiments, the MHC protein subunit does not include a transmembrane domain.

  Peptide (P) can be about 6-12 amino acids long, for example, about 8-10 amino acids for a complex with a class I MHC protein. The peptide can be about 6-20 amino acids in length for a complex with a Class II MHC protein, for example, about 10-18 amino acids. Peptides can have sequences from a wide variety of proteins. The peptide can be a T cell epitope. Epitope sequences from several antigens are known in the art. Alternatively, the epitope sequence can be obtained by isolating and sequencing the peptide bound to the native MHC protein, by synthesizing a series of peptides from the target sequence, and then assaying for T cells reactive to different peptides. , Or by generating a series of binding complexes with different peptides and quantifying T cell binding. Fragment preparation, sequence identification, and minimal sequence identification are described in US Pat. No. 5,019,384 and references cited therein. Peptides can be prepared in various ways. Conveniently, they can be synthesized by conventional techniques using an automatic synthesizer, or they can be synthesized manually. Alternatively, a DNA sequence encoding a particular peptide can be prepared, cloned and expressed to provide the desired peptide. In this case, methionine may be the first amino acid. In addition, the peptides can be purified by recombinant methods, such as fusion to a protein that is one of a specific binding pair, which allows purification of the fusion protein by an affinity reagent, and then at a normally engineered site. It can be produced by proteolytic cleavage to yield the desired peptide (see, for example, Driscoll et al. (1993) J. Mol. Bio. 232: 342-350). Peptides can also be isolated from natural sources and purified by known techniques including, for example, chromatography on ion exchange materials, separation by size, immunoaffinity chromatography and electrophoresis.

  In some embodiments, the α-subunit and β-subunit are produced separately and associated in vitro to form a stable heteroduplex complex (eg, Altman et al. (1993) ) Or Garboczi et al. (1992)). In some embodiments, the α-subunit and β-subunit are expressed together in a single cell. In some embodiments, a single molecule having an α-subunit and a β-subunit is used. For example, single-chain heterodimers can be created by fusing the two subunits together using a short peptide linker, such as a 15-25 amino acid peptide or linker (see, eg, Bedzyk et al. (1990). Year) J. Biol. Chem. 265: 18615). Soluble heterodimers can also be produced by isolation of a native heterodimer and cleavage with a protease, such as papain, to produce a soluble product.

  Soluble subunits can be expressed independently from the DNA construct encoding the truncated protein. For expression, the DNA sequence may be inserted into a suitable expression vector, wherein the native transcription initiation region may be used or associated with an exogenous transcription initiation region, for example, a gene in a chromosome that is normally present. Promoters other than promoters can be used. The promoter can be introduced in vitro by recombinant methods, or as a result of homologous integration of the sequence into the chromosome. Transcription initiation regions are known for a wide variety of expression hosts. Expression hosts may be prokaryotic or eukaryotic, especially E. coli. coli, B.E. subtilis, mammalian cells such as CHO cells, COS cells, monkey kidney cells, lymphoid cells, human cell lines, and the like.

  The subunit can be expressed in a suitable host cell and can be solubilized if necessary. The two subunits may be combined with a peptide and capable of folding in vitro to form a stable heterodimeric complex with an intrachain disulfide-linked domain. The peptide can be included in an initial folding reaction or can be added to the empty heterodimer in a later step. The MHC binding site may be peptide-free prior to addition of the peptide. The exception is when it is desired to label T cells with natural peptide-MHC complexes, such as those that may be on the surface of cells that are targets of an autoimmune attack. MHC heterodimers bind peptides in a groove formed by two membrane distal domains, either α2 and α1 for class I, or α1 and β1 for class II. Conditions that permit folding and association of subunits and peptides are known in the art (see, for example, Altman et al. (1993) and Garboczi et al. (1992)). As an example of an acceptable condition, roughly equimolar amounts of solubilized α and β subunits are mixed in a solution of urea. Refolding is initiated by dilution or dialysis into a buffer solution without urea. The peptide is loaded into the empty class II heterodimer at about pH 5-5.5 for about 1-3 days, followed by neutralization, concentration and buffer exchange. However, it will be readily appreciated by those skilled in the art that the specific folding conditions are not critical to the practice of the present invention.

  In some embodiments, the monomer complex (α-β-P) can be multimerized. For example, multimers can be formed by attaching a monomer to a multivalent entity via a specific attachment site on the α or β subunit. In some embodiments, the multimers are formed by chemical cross-linking of monomers. Several reagents capable of cross-linking proteins are known in the art, including azidobenzoyl hydrazide, N- [4- (p-azidosalicylamino) butyl] -3 '-[ 2′-pyridyldithio] propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipate, disuccinimidyl tartrate, N-γ-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl- 4-azidobenzoate, N-succinimidyl [4-azidophenyl] -1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl] aminobenzoate, glutaraldehyde, formaldehyde and succinimidyl 4- [N-maleimidomethyl ] Cyclohexane-1 Including but carboxylates without limitation. The attachment site for binding to a multivalent entity may be naturally occurring or introduced via genetic engineering. The sites can be members of a specific binding pair, or modified to provide members of a specific binding pair, wherein the complementary pair has a variety of specific binding sites. Binding to a complementary binding member can be a chemical reaction, epitope-receptor binding or hapten-receptor binding, where the hapten is linked to a subunit chain. In a preferred embodiment, one of these subunits is fused to an amino acid sequence that provides a recognition site for the modifying enzyme. This recognition sequence is usually fused proximal to the carboxy terminus of one of these subunits to avoid potential interference at the antigenic peptide binding site. Conveniently, the expression cassette includes a sequence encoding a recognition site.

Modified enzymes of interest include BirA, various glycosylases, farnesyl protein transferases, protein kinases, and the like. The subunit can be reacted with the modifying enzyme at any convenient time, usually after formation of the monomer. Groups introduced by the modifying enzymes, e.g., biotin, sugar, phosphate, farnesyl, and the like, may be members of a complementary binding pair, or further modified to provide a member of a complementary binding pair, e.g., chemical cross-linking, biotinylation. Provide a unique site. An alternative strategy is to introduce an unpaired cysteine residue into the subunit, thereby introducing a chemically reactive site unique to the bond. The attachment site can also be a naturally occurring or introduced epitope, and the multivalent binding partner is an antibody, eg, IgG, IgM, and the like. Any modifications are at sites that do not interfere with binding, eg, near the C-terminus. An example of multimer formation is the introduction of a recognition sequence for the enzyme BirA, which catalyzes the biotinylation of a protein substrate. The monomer with the biotinylated subunit is then conjugated to a multivalent binding partner, such as streptavidin or avidin, to which biotin binds with extremely high affinity. Streptavidin has a valency of 4 and provides multimers of (α-β-P) ₄ . The multivalent binding partner may be free in solution or attached to an insoluble support. Examples of suitable insoluble supports include beads, eg, magnetic beads, membranes, and microtiter plates. These are typically made of glass, plastic (eg, polystyrene), polysaccharide, nylon or nitrocellulose. Attachment to an insoluble support is useful when the binding complex is used for T cell separation.
B cell receptor antigen

  B cells, eg, a subpopulation of B cells or a subset of B cells, can recognize antigen through the B cell receptor (BCR). Tetramers with subunits of the B cell receptor antigen can be used to identify and study these B cells or B cell subsets. B cell receptor antigens include any antigen having an epitope capable of inducing an immune response through the BCR. For example, a B cell receptor antigen may be an antigen that has been used to immunize a subject. B cell receptor antigens can be made into tetramers using the small linear peptide portion of the B cell receptor antigen, where the peptide portion contains a BCR epitope. The small linearized peptide moiety can then be expressed at the site of biotinylation in the expression vector and purified as a monomer used in the tetramer. The monomer with the biotinylated subunit can then be conjugated to a multivalent binding partner, such as streptavidin or avidin, to which biotin binds with extremely high affinity. Streptavidin has a valency of four and provides a tetramer of B cell receptor antigen. The resulting tetramer can be purified using a size exclusion column.

Cells In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate is a cell. For example, the affinity moiety can be an intact cell, a cell treated with a compound (eg, a drug), a fixed cell, a lysed cell, or any combination thereof. In some embodiments, the affinity moiety is a single cell. For example, the affinity portion of an affinity-oligonucleotide conjugate can be a T cell or a B cell. In some embodiments, the affinity moiety is a plurality of cells. In some embodiments, the affinity moiety is a T cell. In some embodiments, the affinity moiety is a B cell. In some embodiments, the affinity moiety is an antigen presenting cell (APC). In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate is a particular cell type or cell subset. For example, the affinity portion of the affinity-oligonucleotide conjugate can be a CD4 ⁺ T cell or a CD8 ⁺ T cell. For example, the affinity portion of an affinity-oligonucleotide conjugate can be a T cell that contains a TCR that specifically recognizes a particular antigen. For example, the affinity portion of an affinity-oligonucleotide conjugate can be a T cell that contains a TCR that specifically recognizes a particular MHC-peptide complex. In some embodiments, the affinity moiety is a cell.

Small Molecules In some embodiments, the affinity portion of the affinity-oligonucleotide conjugate is a small molecule, such as a drug. For example, a small molecule can be a macrocycle, inhibitor, drug or chemical compound. In some embodiments, a small molecule comprises no more than 5 hydrogen bond donors. In some embodiments, a small molecule comprises no more than 10 hydrogen bond acceptors. In some embodiments, small molecules have a molecular weight of 500 Daltons or less. In some embodiments, a small molecule has a molecular weight of about 180-500 daltons. In some embodiments, the small molecule comprises an octanol-water partition coefficient log P of 5 or less. In some embodiments, small molecules have a partition coefficient log P between -0.4 and 5.6. In some embodiments, small molecules have a molar refractive index between 40 and 130. In some embodiments, a small molecule contains about 20 to about 70 atoms. In some embodiments, the small molecule has a polar surface area of 140 Angstroms ² or less.

Nucleic acids In some embodiments, the affinity moiety is a polymeric form of ribonucleotides and / or deoxyribonucleotides (adenine, guanine, thymine or cytosine), eg, DNA or RNA (eg, mRNA). DNA includes linear DNA molecules (eg, restriction fragments), double-stranded DNA found in viruses, plasmids, and chromosomes. In some embodiments, the polynucleotide affinity portion is a single-stranded, double-stranded, small interfering RNA (siRNA), messenger RNA (mRNA), transfer RNA (tRNA), chromosome, gene, non-coding genomic sequence. Genomic DNA (eg, fragmented genomic DNA), purified polynucleotide, isolated polynucleotide, hybridized polynucleotide, transcription factor binding site, mitochondrial DNA, ribosomal RNA, eukaryotic polynucleotide, Prokaryotic polynucleotides, synthesized polynucleotides, ligated polynucleotides, recombinant polynucleotides, polynucleotides containing nucleic acid analogs, methylated polynucleotides, demethylated polynucleotides, any fragments thereof, or Is any combination. In some embodiments, the affinity moiety is a polynucleotide that includes a double-stranded region and a non-double-stranded end (eg, a 5 ′ or 3 ′ overhang region). In some embodiments, the affinity moiety is a recombinant polynucleotide. In some embodiments, the affinity moiety is a heterologous polynucleotide. For example, an affinity moiety can include a polynucleotide (eg, a polynucleotide heterologous to an organism) produced in bacteria (eg, E. coli), yeast, mammals, or insect cells. In some embodiments, the affinity moiety is a polynucleotide comprising a mutation, insertion, deletion or polymorphism.

  In some embodiments, the affinity moiety is an aptamer. Aptamers are isolated nucleic acid molecules that bind with high specificity and affinity to a target analyte, such as a protein. Aptamers are three-dimensional structures that are maintained in a particular conformation (s) that provide chemical contact to specifically bind a given target. Aptamers are nucleic acid-based molecules, but there are fundamental differences between aptamers and other nucleic acid molecules such as genes and mRNA. In the latter, the nucleic acid structure encodes information via its linear base sequence, and this sequence is therefore important for the function of information storage. In stark contrast, aptamer function based on specific binding of the target molecule is not entirely dependent on conserved linear base sequences (non-coding sequences), but rather on specific secondary / tertiary / quaternary structures. Depends on. Any coding potential that an aptamer may have is entirely random and plays no role in binding the aptamer to its cognate target. Aptamers must also be distinguished from naturally occurring nucleic acid sequences that bind to a particular protein. These latter sequences are naturally occurring nucleic acids embedded in the genome of an organism that bind to specialized subgroups of proteins involved in the transcription, translation and transport of naturally occurring nucleic acids (eg, nucleic acid binding proteins). Array Aptamers, on the other hand, are short, isolated, non-naturally occurring nucleic acid molecules. Aptamers that bind to nucleic acid binding proteins can be identified, but in most cases, such aptamers have little or no sequence identity to the sequence actually recognized by the nucleic acid binding protein. More importantly, aptamers can bind to virtually any protein (beyond nucleic acid binding proteins) as well as almost any target of interest, including small molecules, carbohydrates, peptides, and the like. For most targets, even proteins, there is no naturally occurring nucleic acid sequence to which it binds. For targets having such sequences, eg, nucleic acid binding proteins, such sequences differ from aptamers as a result of the relatively low binding affinity actually used, as compared to tightly binding aptamers. Aptamers can specifically bind to selected targets and modulate target activity or binding interactions, e.g., via binding, aptamers block their ability to function. obtain. The functional property of specific binding to a target is an intrinsic property of the aptamer. Typical aptamers are 6-35 kDa (20-100 nucleotides) in size and can bind to their targets with micromolar to sub-nanomolar affinities to distinguish closely related targets (eg, aptamers Can selectively bind related proteins from the same gene family). Aptamers can use commonly found intermolecular interactions, such as hydrogen bonding, electrostatic complementarity, hydrophobic contacts, and steric exclusion to bind to specific targets. Aptamers have several desirable characteristics for use as therapeutics and diagnostics, including high specificity and affinity, low immunogenicity, biological efficacy, and excellent pharmacokinetic properties. Aptamers can include molecular stem and loop structures (eg, hairpin loop structures) formed from the hybridization of covalently linked complementary polynucleotides. The stem contains the hybridized polynucleotide, and the loop is the region that covalently joins two complementary polynucleotides.

In some embodiments, the affinity moiety is a plurality of affinity moieties, eg, a mixture or library of affinity moieties. In some embodiments, the affinity moiety is a plurality of different affinity moieties. For example, the affinity moiety may comprise a plurality of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 , 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000 , 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000 or 30,000 affinity moieties.
Affinity-oligonucleotide portion of oligonucleotide conjugate

  The oligonucleotide portion of the affinity-oligonucleotide conjugate is a nucleic acid coupled to the affinity portion of the affinity-oligonucleotide conjugate. In some embodiments, the oligonucleotide portion of the tetramer-oligonucleotide conjugate is a nucleic acid coupled to the affinity portion of the tetramer-oligonucleotide conjugate. In some embodiments, the oligonucleotide portion of the tetramer-oligonucleotide conjugate is a nucleic acid coupled to one or more core elements of the tetramer complex. For example, a nucleic acid may be conjugated to a monomeric or tetrameric streptavidin core. Exemplary schematics of tetramer-oligonucleotide conjugates are shown in FIGS. 10A, 10B, 13, 14, and 18. In some embodiments, the oligonucleotide is directly coupled to the affinity moiety. In some embodiments, the oligonucleotide is indirectly coupled to an affinity moiety. In some embodiments, the oligonucleotide is non-covalently coupled to the affinity moiety. In some embodiments, the oligonucleotide is covalently coupled to the affinity moiety. In some embodiments, the oligonucleotide is a synthetic oligonucleotide. In a preferred embodiment, the oligonucleotide does not substantially interact directly with the target analyte of the affinity moiety.

  Oligonucleotides coupled to the affinity portion of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate may include one or more barcode sequences. For example, an oligonucleotide coupled to an affinity portion of an affinity-oligonucleotide or tetramer-oligonucleotide conjugate may include an antigen ID (AID) sequence and an antigen molecule barcode (AMB) sequence. The oligonucleotide may include an antigen ID (AID) sequence, a fusion sequence, a primer site, a molecular barcode sequence, a constant sequence, or any combination thereof.

  Oligonucleotides may contain chemical modifications that allow conjugation of the affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate to the affinity moiety (eg, amine, thiol or biotin). it can.

  Oligonucleotides can constitute multiple oligonucleotides. The plurality of oligonucleotides can be composed of a plurality of affinity-oligonucleotide conjugates or a plurality of tetramer-oligonucleotide conjugates. For example, the oligonucleotide may comprise a plurality of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000 or 30,000 oligonucleotides may be constituted. For example, a plurality of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1, 000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, The 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, or 30,000 oligonucleotides comprise a plurality of at least about 2,3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1,000, 2,000, 3,000, 4, 000, 5,000, 6,000, 7 000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, It can be constituted by a 20,000, 25,000 or 30,000 affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate.

The oligonucleotide may include an oligonucleotide barcode sequence, an oligonucleotide fusion sequence, an oligonucleotide primer binding sequence, an oligonucleotide constant sequence, or any combination thereof.
Oligonucleotide antigen ID (AID) sequence

  The oligonucleotide may comprise an oligonucleotide antigen barcode sequence or its complement. The oligonucleotide antigen barcode may allow for the identification of an affinity-oligonucleotide or tetrameric oligonucleotide complex comprising the oligonucleotide antigen barcode. The oligonucleotide antigen barcode may allow identification of the affinity moiety to which the oligonucleotide antigen barcode is attached. Oligonucleotide antigen barcodes can be used to identify affinity moieties from multiple different affinity moieties that bind to different target analytes. The oligonucleotide antigen barcode may be barcoded exclusively to the affinity-oligonucleotide complex or exclusively to the tetramer-oligonucleotide complex. The oligonucleotide antigen barcode can be barcoded exclusively on the affinity moiety. Thus, the oligonucleotide antigen barcode sequence can be barcoded to a specific affinity moiety.

  The oligonucleotide antigen barcode can be a unique barcode sequence. For example, any one of the plurality of oligonucleotide antigen barcodes can be a unique barcode sequence. The number of different antigenic barcode sequences that are theoretically possible can depend directly on the length of the barcode sequence. For example, if a DNA barcode with randomly assembled adenine, thymidine, guanosine and cytidine nucleotides can be used, the theoretical maximum number of possible barcode sequences is 1,048,576 for a length of 10 nucleotides. It can be 1,073,741,824 for a length of 15 nucleotides. The oligonucleotide antigen barcode sequence has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 , 45, or 50 or more contiguous nucleotide sequences. Oligonucleotides can include two or more oligonucleotide antigen barcode sequences or their complements. Oligonucleotide antigen barcode sequences can include randomly assembled sequences of nucleotides. The oligonucleotide antigen barcode sequence can be a degenerate sequence. The oligonucleotide antigen barcode sequence can be a known sequence. The oligonucleotide antigen barcode sequence can be a predefined sequence. In a preferred embodiment, the oligonucleotide antigen barcode sequence comprises an oligonucleotide antigen barcode or its complement to identify one of a plurality of different affinity moieties that interact with different target analytes. A known unique sequence that is barcoded into the affinity moiety to which it is coupled so that the containing signal (eg, a sequence readout) can be used.

  For example, an oligonucleotide coupled to an affinity portion of an affinity-oligonucleotide conjugate can include a barcode that is an antigen ID (AID) sequence. The AID sequence can be barcoded on the affinity portion of the affinity-oligonucleotide conjugate. The AID sequence can be barcoded on the antigen targeted by the affinity moiety. The AID sequence can be used to identify the affinity portion of the affinity-oligonucleotide conjugate and / or the antigen that the affinity portion targets. For example, the AID sequence can be barcoded on the antibody of the antibody-oligonucleotide conjugate. For example, the AID sequence can be barcoded to the antigen targeted by the antibody of the antibody-oligonucleotide conjugate. For example, AID sequences can be used to immunophenotype cells. For example, the AID sequence can be barcoded on a peptide of the MHC-peptide complex.

  The AID sequence may be unique for each antigen targeted by the affinity portion of the affinity-oligonucleotide conjugate. The AID sequence may be unique to the affinity portion of the affinity-oligonucleotide conjugate. For example, the AID sequence may be unique for each antibody that specifically binds to a different target antigen on the cell. In some embodiments, the AID sequence is a defined sequence. In some embodiments, the AID sequence is a known sequence. The AID sequence for each oligonucleotide can be determined by sequencing the oligonucleotide or the amplification product of the oligonucleotide, for example, by next generation sequencing.

Oligonucleotide molecule barcode sequence The oligonucleotide may comprise an oligonucleotide molecule barcode sequence or its complement. The oligonucleotide barcode may allow for the identification of the molecules of the affinity-oligonucleotide complex containing the oligonucleotide barcode. Oligonucleotide molecule barcodes can be barcoded exclusively to the molecules of the affinity-oligonucleotide complex. The oligonucleotide molecule barcode can be barcoded exclusively to the molecule of the affinity moiety. Thus, the oligonucleotide molecule barcode sequence can be barcoded to a specific molecule of the affinity moiety.

  The oligonucleotide molecule barcode can be a unique barcode sequence. For example, any one of the plurality of oligonucleotide molecule barcodes may be a unique barcode sequence. The number of different molecular barcode sequences that are theoretically possible can depend directly on the length of the barcode sequence. For example, if a DNA barcode with randomly assembled adenine, thymidine, guanosine and cytidine nucleotides can be used, the theoretical maximum number of possible barcode sequences is 1,048,576 for a length of 10 nucleotides. It can be 1,073,741,824 for a length of 15 nucleotides. The oligonucleotide molecule barcode sequence has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40. , 45, or 50 or more contiguous nucleotide sequences. Oligonucleotides can include two or more oligonucleotide molecule barcode sequences or their complements. An oligonucleotide molecule barcode sequence can include a randomly assembled sequence of nucleotides. The oligonucleotide molecule barcode sequence can be a degenerate sequence. The oligonucleotide molecule barcode sequence can be a known sequence. The oligonucleotide molecule barcode sequence can be a predefined sequence. In a preferred embodiment, the oligonucleotide molecule barcode sequence is a unique sequence and can be used to identify one of a plurality of affinity submolecules that interacts with a target analyte.

For example, an oligonucleotide coupled to an affinity portion of an affinity-oligonucleotide conjugate can include a barcode that is an antigen molecule barcode (AMB) sequence. The antigen molecule barcode (AMB) sequence may be unique for each oligonucleotide molecule of the affinity-oligonucleotide conjugate. The AMB sequence may allow counting of the number of oligonucleotide molecules of the affinity-oligonucleotide conjugate bound to an antigen, such as an antigen of an individual cell in a vessel, eg, an emulsion droplet. The AMB sequence for each oligonucleotide can be determined by sequencing the oligonucleotide or the amplification product of the oligonucleotide, for example, by next generation sequencing.
Oligonucleotide fusion sequence

  Oligonucleotides coupled to the affinity portion of the affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate may include a fusion sequence. The fusion sequence can be an affinity-oligonucleotide conjugate, e.g., a cell surface bound affinity-oligonucleotide conjugate, or a tetramer-oligonucleotide conjugate, e.g., a TCR or BCR-bound tetramer-oligonucleotide conjugate. May allow PCR extension of a droplet-specific barcode sequence onto an oligonucleotide. The fusion sequence of each oligonucleotide of the plurality of oligonucleotides may be identical. The fusion sequence can include a sequence that is complementary to the sequence of the droplet barcode. In some embodiments, the fusion sequence is located at the end of the oligonucleotide. In some embodiments, the terminal fusion sequence of the oligonucleotide is not directly conjugated to the affinity portion of the antibody-oligonucleotide or tetramer-oligonucleotide conjugate. In some embodiments, the fusion sequence at the end of the oligonucleotide comprises a free end.

  The fusion sequence can include a region that is complementary to a region of the 3'-tagging polynucleotide, e.g., a polynucleotide that includes a Bessel barcode. The fusion sequence can include a region that is complementary to the complement of a polynucleotide, eg, a region of the polynucleotide that includes a Bessel barcode. For example, a fusion sequence can include a 3 'region complementary to a 3' tagging polynucleotide or a complement thereof, including a barcode, such as a Bessel barcode, for example, a 3 'terminal region.

  A 3 'tagging polynucleotide is a polynucleotide used to add a nucleic acid to the 3' end of a target polynucleotide, such as an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate oligonucleotide. Good. A 3 'tagging polynucleotide is a polynucleotide used as a template to add nucleic acid to the 3' end of a target polynucleotide, such as an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate oligonucleotide. May be. The 3 'tagging polynucleotide can be a polynucleotide that hybridizes to the 3' end of a target polynucleotide, such as an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate oligonucleotide. The 3 ′ tagging polynucleotide has a 3 ′ region, eg, a 3 ′ terminal region, that hybridizes to the 3 ′ end of a target polynucleotide, such as an oligonucleotide of an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate. It may be a contained polynucleotide.

  In some embodiments, the 3 'tagging polynucleotide is a Bessel bar-encoding polynucleotide. The vessel barcode can be added to the oligonucleotide of the affinity-oligonucleotide conjugate or the tetramer-oligonucleotide conjugate. For example, a vessel barcode can be hybridized to an affinity-oligonucleotide or tetramer-oligonucleotide conjugate oligonucleotide. The Vessel bar-encoding polynucleotide can include a 3 'region, such as a 3' end region, that hybridizes to the 3 'end of the oligonucleotide of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate. .

  In some embodiments, the 3 'tagging polynucleotide is an amplification product. In some embodiments, the 3 'tagging polynucleotide is an amplification product that results from a single molecule. In some embodiments, the 3'tagging polynucleotide is an amplification product of a Bessel bar-encoding polynucleotide. In some embodiments, the 3'tagging polynucleotide is an amplification product resulting from a single Vessel barcoding polynucleotide. The region 5 'to the 3' region that hybridizes to the 3 'end of the oligonucleotide of the affinity-oligonucleotide conjugate can include a region complementary to the primer or its complement. The region 5 'to the 3' region that hybridizes to the 3 'end of the oligonucleotide of the affinity-oligonucleotide conjugate is the oligonucleotide of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate. Can comprise a region complementary to a primer that can be used to amplify For example, a first complementary to a region 5 'to a 3' region that hybridizes to the 3 'end of an affinity-oligonucleotide conjugate, a tetramer-oligonucleotide conjugate oligonucleotide or its complement. A primer set comprising an affinity-oligonucleotide conjugate or a second primer complementary to the primer site of the oligonucleotide of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate. Alternatively, it can be used to amplify the oligonucleotide of a tetramer-oligonucleotide conjugate.

  The region 5 'to the 3' region that hybridizes to the 3 'end of the oligonucleotide of the affinity-oligonucleotide conjugate is the primer used to amplify the Bessel bar-encoded polynucleotide or its complement. May comprise a region complementary to the other.

The oligonucleotide fusion sequence has at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, It can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous nucleotides. The oligonucleotide fusion sequence can be a sequence of known length. The oligonucleotide fusion sequence can be a known sequence. The oligonucleotide fusion sequence can be a predefined sequence. The oligonucleotide fusion sequence can be an unknown sequence of known length. The oligonucleotide fusion sequence can be a known sequence of known length.
Oligonucleotide constant sequence

  Oligonucleotides coupled to the affinity portion of the affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate can include a constant sequence. This constant sequence is optional. The constant sequence of each oligonucleotide of the plurality of affinity-oligonucleotide or tetramer-oligonucleotide conjugates may be identical.

  Oligonucleotide constant sequences can be used to increase the length of the oligonucleotide or to separate one or more of the oligonucleotide barcodes, oligonucleotide fusions and oligonucleotide primer binding sites from each other. In some embodiments, the oligonucleotide does not include an oligonucleotide constant sequence. For example, an oligonucleotide can be coupled to an affinity moiety at the end of the oligonucleotide that contains the oligonucleotide primer binding site.

In some embodiments, the oligonucleotide constant sequence is attached to an affinity portion of an affinity-oligonucleotide or tetramer-oligonucleotide complex. In some embodiments, the oligonucleotide constant is located upstream of the oligonucleotide primer binding sequence. For example, the oligonucleotide constant sequence can be located 5 'to the oligonucleotide primer binding sequence. In some embodiments, the oligonucleotide constant is located downstream of the oligonucleotide primer binding sequence. For example, the oligonucleotide constant sequence can be located 3 'to the oligonucleotide primer binding sequence. In some embodiments, the oligonucleotide constant is located upstream of the oligonucleotide barcode. For example, the oligonucleotide constant sequence can be located 5 'to the oligonucleotide barcode. In some embodiments, the oligonucleotide constant is located downstream of the oligonucleotide barcode. For example, the oligonucleotide constant sequence can be located 3 'to the oligonucleotide barcode. In some embodiments, the oligonucleotide constant is located upstream of the oligonucleotide fusion sequence. For example, an oligonucleotide constant sequence can be located 5 'to an oligonucleotide fusion sequence.
In some embodiments, the oligonucleotide constant sequence is sandwiched between the oligonucleotide primer binding sequence and the affinity portion of the affinity-oligonucleotide or tetramer-oligonucleotide complex. For example, an oligonucleotide constant sequence can be located 5 'to the oligonucleotide primer binding sequence and can be attached to an affinity portion of the oligonucleotide. In some embodiments, the oligonucleotide constant sequence is flanked between the oligonucleotide primer binding sequence and the oligonucleotide barcode. For example, an oligonucleotide constant sequence can be located 3 'to the oligonucleotide primer binding sequence and 5' to the oligonucleotide barcode. In some embodiments, the oligonucleotide constant is sandwiched between the oligonucleotide fusion sequence and the oligonucleotide barcode. For example, an oligonucleotide constant sequence can be located 3 'to the oligonucleotide barcode and 5' to the oligonucleotide fusion sequence.
The oligonucleotide constant sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30. , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 250, 300, 400, 500 or more contiguous nucleotides. Oligonucleotide constant sequences can include non-random sequences of nucleotides. Oligonucleotide constant sequences may be sequences of known length. The oligonucleotide constant sequence may be a known sequence. The oligonucleotide constant sequence may be a predefined sequence. The oligonucleotide constant sequence may be an unknown sequence of known length. The oligonucleotide constant sequence can be a known sequence of known length.
Oligonucleotide primer binding site

  The oligonucleotide coupled to the affinity portion of the affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate can include a primer site. A primer site can include a sequence that is complementary to a primer, such as an amplification primer. Oligonucleotide primer binding sequences can be used as primer binding sites for reactions such as amplification or sequencing. The oligonucleotide primer binding sequence may be a first primer binding sequence for a pair of primers used for a reaction such as amplification or sequencing. For example, the oligonucleotide primer binding sequence may be a forward primer binding site. For example, the oligonucleotide primer binding site may be a reverse primer binding site. For example, the oligonucleotide primer binding site may be a forward primer binding site, and the primer binding sequence of the Vessel bar-coded polynucleotide attached to the oligonucleotide may be a reverse primer binding sequence. In some embodiments, the oligonucleotide primer binding sequence is a universal primer binding sequence.

  A melting temperature that differs by no more than 6, 5, 4, 3, 2, or 1 degree Celsius from the oligonucleotide primer binding sequence and the primer binding sequence of the polynucleotide attached to the oligonucleotide (eg, of a Bessel bar coded polynucleotide). May be included. The nucleotide sequence of the oligonucleotide primer binding sequence, and the nucleotide sequence of the primer binding sequence of the polynucleotide attached to the oligonucleotide, the polynucleotide that hybridizes to the oligonucleotide primer binding sequence, the polynucleotide attached to the oligonucleotide It may be different so as not to hybridize to the primer binding sequence. The nucleotide sequence of the oligonucleotide primer binding sequence and the nucleotide sequence of the primer binding sequence of the polynucleotide attached to the oligonucleotide are a polynucleotide that hybridizes to the primer binding sequence of the polynucleotide attached to the oligonucleotide, It may be different so as not to hybridize to the primer binding sequence.

Arrangement of Oligonucleotide Elements The oligonucleotides can be arranged in an order such that the oligonucleotide fusion sequence is located at one end of the oligonucleotide. Oligonucleotides can be arranged in an order to include an oligonucleotide barcode upstream of the oligonucleotide fusion sequence. Oligonucleotides can be arranged in an order to include an oligonucleotide primer binding sequence upstream of the oligonucleotide barcode. The oligonucleotides can be arranged in an order such that the oligonucleotide constant sequence is located upstream or downstream of the oligonucleotide primer binding sequence. The oligonucleotides can be arranged in an order such that the oligonucleotide constant sequence is located upstream of the oligonucleotide barcode sequence. The oligonucleotides can be arranged in an order such that the oligonucleotide constant sequence is located at one end of the oligonucleotide, eg, at the end of the oligonucleotide that does not include the oligonucleotide fusion sequence. For example, the oligonucleotides can be arranged in the following order: an oligonucleotide fusion sequence, an oligonucleotide barcode sequence, an oligonucleotide primer binding sequence, and an oligonucleotide constant sequence. For example, the oligonucleotides can be arranged in the order of an oligonucleotide fusion sequence, an oligonucleotide barcode sequence, an oligonucleotide primer binding sequence, and an oligonucleotide constant sequence, toward or from the affinity portion. For example, the oligonucleotides may be arranged from the 5 'end to the 3' end or from the 3 'end to the 5' end in the order of an oligonucleotide fusion sequence, an oligonucleotide barcode sequence, an oligonucleotide primer binding sequence, and an oligonucleotide constant sequence. Can be done. For example, oligonucleotides can be attached to the 5 'terminal oligonucleotide fusion sequence, a unique oligonucleotide barcode sequence, a reverse oligonucleotide primer binding sequence, and an affinity moiety (eg, via a primary amine group attached to the 3' end). )) The attached 3 'oligonucleotide constant sequence may be included in that order. For example, an oligonucleotide attached to an affinity moiety can be arranged in the order of an oligonucleotide fusion sequence, an oligonucleotide barcode sequence, an oligonucleotide constant sequence, and an oligonucleotide primer binding site sequence that propagates toward the affinity moiety. .

  The oligonucleotides can be arranged in an order such that the fusion sequence is located at one end of the oligonucleotide. The oligonucleotides can be arranged in an order such that the fusion sequence is located downstream of the AID sequence. The oligonucleotides can be arranged in an order such that the fusion sequence is located downstream of the AMB sequence. The oligonucleotides can be arranged in an order such that the fusion sequence is located downstream of the constant sequence. The oligonucleotides can be arranged in an order such that the fusion sequence is located downstream of the primer site.

  The oligonucleotides can be arranged in an order such that the primer sites are located at one end of the oligonucleotide. The oligonucleotides can be arranged in an order such that the primer sites are located upstream of the AID sequence. The oligonucleotides can be arranged in an order such that the primer sites are located upstream of the AMB sequence. The oligonucleotides can be arranged in an order such that the primer sites are located upstream of the constant sequence. The oligonucleotides may be arranged in an order such that the primer sites are located upstream of the fusion sequence.

  The oligonucleotides can be arranged in an order to include the AID sequence upstream of the fusion sequence. Oligonucleotides can be arranged in an order to include the AID sequence downstream of the primer site. The AID sequence may be located upstream or downstream of the AMB sequence. The AID sequence may be located upstream or downstream of the constant sequence. Oligonucleotides can be arranged in an order to include the AID sequence between the fusion sequence and the primer site.

  Oligonucleotides can be arranged in an order to include the AMB sequence upstream of the fusion sequence. Oligonucleotides can be arranged in an order to include the AMB sequence downstream of the primer site. The AMB sequence may be located upstream or downstream of the AID sequence. The AMB sequence may be located upstream or downstream of the constant sequence. Oligonucleotides can be arranged in an order to include the AMB sequence between the fusion sequence and the primer site.

  Oligonucleotides can be arranged in an order to include the constant sequence upstream of the fusion sequence. Oligonucleotides can be arranged in an order to include the constant sequence downstream of the primer site. The constant sequence may be located upstream or downstream of the AID sequence. The constant sequence may be located upstream or downstream of the AMB sequence. Oligonucleotides can be arranged in an order to include a constant sequence between the fusion sequence and the primer site.

  The oligonucleotides may be arranged in an order such that the AMB and / or AID sequences are not located at one end of the oligonucleotide, eg, at the end of the oligonucleotide containing the fusion sequence or primer site. For example, the oligonucleotides can be arranged in the following order: fusion sequence, AID sequence, AMB sequence and primer site. For example, the oligonucleotides can be arranged in the following order: fusion sequence, AMB sequence, AID sequence and primer sites.

  For example, the oligonucleotides can be arranged in the following order: fusion sequence, AID sequence, AMB sequence, constant sequence, and primer site. For example, the oligonucleotides can be arranged in the following order: fusion sequence, AMB sequence, AID sequence, constant sequence and primer sites. For example, the oligonucleotides can be arranged in the following order: fusion sequence, constant sequence, AID sequence, AMB sequence and primer sites. For example, the oligonucleotides can be arranged in the following order: fusion sequence, constant sequence, AMB sequence, AID sequence and primer site. For example, the oligonucleotides can be arranged in the following order: fusion sequence, AID sequence, constant sequence, AMB sequence and primer sites. For example, the oligonucleotides can be arranged in the following order: fusion sequence, AMB sequence, constant sequence, AID sequence and primer sites.

For example, the oligonucleotides can be arranged in the following order: fusion sequence, AID sequence, AMB sequence, constant sequence, and primer sites, which travel toward the affinity portion of the affinity-oligonucleotide conjugate. For example, the oligonucleotide can be arranged from the 5 'end to the 3' end of the oligonucleotide, in the order of fusion sequence, AID sequence, AMB sequence, constant sequence, and primer site. For example, oligonucleotides can be attached to the 5'-end fusion sequence, the AID sequence, the AMB sequence, the constant sequence, and the affinity portion of the affinity-oligonucleotide conjugate (eg, a primary antibody attached to the 3 'end of the oligonucleotide). 3 'primer sites attached (via amine groups) may be included in that order.
Affinity-oligonucleotide conjugate preparation

  The affinity-oligonucleotide or tetramer-oligonucleotide conjugate used in the methods and compositions described herein can be prepared using any convenient method. The affinity moiety can be directly or indirectly coupled to the oligonucleotide (eg, via a linker). The affinity moiety can be covalently (eg, via chemical crosslinking) or non-covalently (eg, via streptavidin-biotin) coupled to the oligonucleotide. Affinity, including various different affinity moieties that can be used, designing oligonucleotides for proximity ligation assays, and coupling such oligonucleotides to affinity moieties to form affinity-oligonucleotide conjugates -The design and preparation of oligonucleotide conjugates has been widely described in the art. The details and principles described in the art can be applied to the design of affinity-oligonucleotide conjugates for use in the methods of the invention (eg, WO2007107743, and US Patent Nos. 7,306,904 and No. 6,878,515).

  A direct coupling reaction between the oligonucleotide and the affinity moiety can be, for example, a functional group (eg, a substituent or a chemical handle), each of which can react with a functional group on the other. ) Can be used. The functional group can be present on the oligonucleotide or the affinity moiety, or can be introduced onto these components (eg, via an oxidation reaction, a reduction reaction, a cleavage reaction, etc.). Methods for producing nucleic acid / polypeptide conjugates have been described (see, eg, US Pat. No. 5,733,523).

The functional groups of the antibodies or polypeptides may be used for coupling to oligonucleotides, carbohydrates, thiol group of an amino acid (HS-), the amino acid of the amine group (H 2 _N-), and a carboxy group of an amino acid Are not limited to these. For example, a carbohydrate structure may be oxidized to an aldehyde and reacted with a H ₂ NNH group containing compound to form the functional group —C ＝ NH—NH—. For example, a thiol group can be reacted with a thiol-reactive group to form a thioether or disulfide. For example, a free thiol group of a protein can be introduced into a protein by thiolation or cleavage of a disulfide in a native cysteine residue. For example, an amino group (eg, at the amino terminus, or the omega amino group of a lysine residue) can be reacted with an electrophilic group (eg, an activated carboxy group) to form an amide group. For example, a carboxy group (eg, the carboxy terminus, or the carboxy group of a diacidic alpha amino acid) can be activated and contacted with an amino group to form an amide group. Other exemplary functional groups include, for example, SPDP, carbodiimide, glutaraldehyde, and the like.

  In an exemplary embodiment, the oligonucleotide is covalently coupled to the affinity moiety using a commercially available kit ("All-in-One Antibody-Oligonucleotide Conjugation Kit"; Solulink, Inc.). For example, first, a 3'-amino-oligonucleotide can be derivatized with sulfo-S-4FB. Second, the affinity moiety can be modified with an S-HyNic group. Third, the HyNic modified affinity moiety can be reacted with a 4FB modified oligonucleotide to obtain a bis-arylhydrazone mediated affinity-oligonucleotide conjugate. Excess 4FB modified oligonucleotide can be further removed via a magnetic affinity matrix. Recovery of the overall affinity moiety is at least about 95%, 96%, 97%, 98%, 99% or 100% free of the HyNic modified affinity moiety and the 4FB modified oligonucleotide. Bis-arylhydrazone linkages can be stable to both heat (eg, 94 ° C.) and pH (eg, 3-10).

  If a linking group is used, such a linker can be selected to provide a covalent or non-covalent attachment of the affinity moiety to the oligonucleotide via the linking group. Various suitable linkers are known in the art. In some embodiments, the linker is at least about 50 or 100 daltons 100 daltons. In some embodiments, the linker is at most about 300; 500; 1,000; 10,000 or 100,000 daltons. The linker may include, at either end, a functional group having a reactive functionality capable of binding to the oligonucleotide. The linker may include, at either end, a functional group having a reactive functionality capable of binding to the affinity moiety. The functional groups can be present on the oligonucleotide, the affinity moiety and / or the linker, or can be introduced onto these components (eg, via an oxidation reaction, a reduction reaction, a cleavage reaction, etc.).

  Exemplary linkers include polymers, aliphatic hydrocarbon chains, unsaturated hydrocarbon chains, polypeptides, polynucleotides, cyclic linkers, acyclic linkers, carbohydrates, ethers, polyamines, and others known in the art. Things included. Exemplary functional groups of the linker include nucleophilic functional groups (eg, amines, amino groups, hydroxy groups, sulfhydryl groups, amino groups, alcohols, thiols and hydrazides), electrophilic functional groups (eg, aldehydes, esters, vinyl ketones) Epoxides, isocyanates and maleimides), and functional groups capable of cycloaddition, capable of forming disulfide bonds, or capable of binding metal. For example, the functional groups of the linker are primary amine, secondary amine, hydroxamic acid, N-hydroxysuccinimidyl ester, N-hydroxysuccinimidyl carbonate, oxycarbonylimidazole, nitrophenyl ester, trifluoroethyl ester, glycidyl ether , Vinyl sulfone, maleimide, azidobenzoyl hydrazide, N- [4- (p-azidosalicylamino) butyl] -3 '-[2'-pyridyldithio] propionamide (N- [4- (p-azidosalicylamino) butyl] -3 '-[2'-pyridyldithio] propionamid)), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl Jil-4-azidobenzoate, N-Sk Nimidyl [4-azidophenyl] -1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl] aminobenzoate, glutaraldehyde, and succinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate , 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like.

  In other embodiments, the affinity-oligonucleotide or tetramer-oligonucleotide conjugate is an affinity-oligonucleotide conjugate, such as producing an affinity moiety in vitro from a vector encoding the affinity moiety. It can be produced using an in vitro protocol that produces a gate or tetramer-oligonucleotide conjugate. Examples of such in vitro protocols of interest include the following: RepA-based protocols (see, eg, Fitzgerald et al., Drug Discov Today (2000) 5: 253-58, and WO9837186), ribosomes. Display-based protocols (e.g., Hanes et al., PNAS (1997) 94: 4937-42; Roberts et al., Curr Opin Chem Biol (1999) June; 3: 268-73; Schaffitzel et al., J Immunol. Methods (1999) December 10; 231: 119-35; and WO9854312).

  Techniques for conjugating nucleic acid molecules to antibodies are well known in the art (eg, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy (Ed. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (edited by Robinson et al., Marcel Deiker, Inc., 2nd edition 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In. Cancer Therapy: A Review, Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., Eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy, Monoclonal Antibodies For Cancer Detection And Therapy (eds. Baldwin et al., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62. Pp 119-58 see. E.g., PCT Publication WO89 / 12624 see also). For example, a nucleic acid molecule can be covalently attached to lysine or cysteine on an antibody, for example, via an N-hydroxysuccinimide ester or maleimide functionality, respectively.

Target antigen The target antigen of the affinity portion can be a nucleic acid molecule or can be proteinaceous, eg, a target protein or peptide. The target antigen can be a compound or composition present on cells in the sample. In some embodiments, the target antigen can be a compound or composition capable of eliciting a cell-mediated immune response (ie, an adaptive immune response), particularly in a mammal such as a human. In some embodiments, the target antigen may be recognized by a T cell in the context of an MHC molecule. Target antigens include, but are not limited to, cells, tissue extracts, tissue or cell lysates, proteins, multiple proteins, peptides, mixtures of peptides, lipids, carbohydrates, sugars, etc., individually or as a mixture. . The target antigen may be characteristic of a disease, such as an infectious disease, an autoimmune disease or cancer. The target antigen can be, for example, a viral antigen, a bacterial antigen, a cancer antigen, and the like.

  In some embodiments, the target antigen is a viral antigen. Viral antigens include, for example, virus coat protein, influenza virus antigen, HIV antigen, hepatitis B antigen or hepatitis C antigen.

  In some embodiments, the target antigen is a cancer antigen (eg, a protein, peptide, or the like) that is exclusively or predominantly expressed or overexpressed by tumor cells or cancer cells such that the antigen is associated with the tumor or cancer. Lipids, carbohydrates, etc.). The cancer antigen can be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen may be (eg, may be a feature of) more than one type of cancer or tumor cancer antigen. For example, a cancer antigen can be expressed by both breast and prostate cancer cells, not expressed at all, or only minimally, by normal, non-tumor or non-cancer cells. The cancer antigen can be a melanoma cancer antigen or a breast cancer antigen. Exemplary cancer antigens include gp100, MART-1, NY-ESO-1, a member of the MAGE family of proteins, such as MAGE-A1, mesothelin, tyrosinase, TRP-1, TRP-2, PMSA, Her-2. And p53.

  The target antigen can be produced naturally, artificially, synthetically or recombinantly. Thus, the target antigen can be a synthetic, recombinant, isolated and / or purified protein, polypeptide or peptide. Methods for making or obtaining such antigens are known in the art. For example, suitable methods for de novo synthesis of polypeptides and proteins (eg, antigenic polypeptides and proteins) are described in Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, Reid, R., Ed., Marcel Dekker, Inc., 2000; Epitope Mapping, Westwood et al., Ed., Oxford University Press, Oxford, United Kingdom, 2000; and US Pat. No. 5,449,752. ing. Also, polypeptides and proteins (eg, antigenic polypeptides and proteins) can be produced recombinantly using nucleic acids encoding the polypeptides or proteins using standard recombinant methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. See year. The nucleotide sequences of many antigens are known in the art and are available from the GenBank database on the National Center for Biotechnology Information (NCBI) website. Further, the antigen may be isolated and / or purified from a source, such as a plant, a bacterium, an insect, a mammal, such as a rat, a human, and the like. Methods for isolation and purification are well-known in the art.

  The antigen may be a free antigen, eg, an unbound antigenic peptide (eg, a free peptide), or an MHC-peptide tetramer or antigenic presented by a bound antigen, eg, a carrier cell pulsed with the peptide. It can be a peptide.

  In some embodiments, the target analyte is a membrane-bound protein. In one embodiment, the membrane-bound protein is the classical type I membrane protein CD4 with a single transmembrane (TM) domain (Carr et al. (1989) J. Biol. Chem. 264: 21286). ９５95). In another embodiment, the membrane-bound protein is the multi-transmembrane G protein-coupled receptor (GPCR) membrane protein GPR77 (Cain and Monk, (2002) J. Biol. Chem. 277: 7165-). 69).

Further exemplary membrane-bound proteins include GPCRs (eg, adrenergic receptors, angiotensin receptors, cholecystokinin receptors, muscarinic acetylcholine receptors, neurotensin receptors, galanin receptors, dopamine receptors, opioid receptors) , Serotonin (erotonin) receptor, somatostatin receptor, etc., ion channels (eg, nicotinic acetylcholine receptor, sodium channel, potassium channel, etc.), receptor tyrosine kinase, receptor serine / threonine kinase, receptor guanylate cyclase , Growth factors and hormone receptors (eg, epidermal growth factor (EGF) receptor) and the like. Mutants or modified variants of membrane-bound proteins can also be used. For example, some single or multiple point mutations in a GPCR retain function and are involved in disease (see, eg, Stadel et al., (1997) Trends in Pharmacological Review 18: 430-37). ).
Single cell characterization, cell polynucleotide barcoding, and chain pairing

  The methods described herein can include characterizing cells using affinity-oligonucleotide conjugates or tetramer-oligonucleotide conjugates. Multiple cells can be contacted with one or more affinity-oligonucleotide conjugates or one or more tetramer-oligonucleotide conjugates. Cells can be washed to remove unbound conjugate. Cells can be isolated as single cells in a vessel. The affinity-oligonucleotide conjugate bound to the isolated cells can be modified to contain a Bessel barcoding sequence, for example, by attaching a Bessel barcoding polynucleotide to the oligonucleotide of the conjugate. it can. The tetramer-oligonucleotide conjugate attached to the isolated cell is modified to contain a Bessel barcode sequence, for example, by attaching a Bessel barcode polynucleotide to the conjugate oligonucleotide. Can be.

  A polynucleotide having a Bessel barcode can also be introduced during formation of the vessel. These Bessel barcoded polynucleotides can have a degenerate barcode such that each oligonucleotide containing a Bessel barcode contains a unique identity code corresponding to the vessel in which they are located.

  The oligonucleotide can be amplified and the amplification product of the reaction can be recovered from the vessel. Amplification products can be PCR-enriched to add next generation sequencing (NGS) tags. Libraries can be sequenced using a high-throughput sequencing platform, followed by analysis of Bessel barcode sequences and / or AID sequences and / or AMB sequences. Since each single cell is isolated in its respective vessel, for each vessel barcode observed twice, the amplified oligonucleotide product sequenced originates in the same vessel and thus has a unique Originated from a single cell. Each AID of the oligonucleotide is barcoded to the affinity portion of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate to which it is attached, and each single cell is in its respective vessel. For each AID observed for a sequence containing the same Bessel barcode, the amplified oligonucleotide product sequenced has a specific affinity bound to a single cell in the same vessel- Originated from oligonucleotide conjugate. For each different AMB individually observed in a set of sequences that all contained the same Bessel barcode, the amplified oligonucleotide product with the AMB in the sequenced set bound to cells in the same vessel. For example, if a single cell vessel is used, a single affinity-oligonucleotide conjugate molecule (compared to another individual AMB) bound to a single cell in the vessel Originated from the oligonucleotide moiety. For each single AMB observed, all amplified oligonucleotide products having the same sequence containing the same Bessel barcode have a single (same) affinity-oligonucleotide conjugate molecule or tetramer- Originated from the oligonucleotide portion of the oligonucleotide conjugate molecule (eg, indicating a PCR overlap or amplicon). Thus, each oligonucleotide observed using a given combination of a particular AMB and a particular Bessel barcode is an affinity-oligonucleotide conjugate bound to a single cell or a 4-cell bound to a single cell. 1 shows a single molecule of a dimer-oligonucleotide conjugate; thus, a given number of sequenced oligonucleotides having such an AMB / Vessel barcode combination (eg, 2, 3, 4, or more) Detection, for example, the number of affinity-oligonucleotide conjugates or tetramer-oligonucleotide conjugates bound to a single cell in the cell population or sample assayed. (Eg, 2, 3, 4, or more). Thus, such a number is the number of molecules on a cell, for example, on a single cell or in a single cell, to which a given affinity portion of an affinity-oligonucleotide conjugate is designed to bind. The number of copies expressed can be indicated.

  In some embodiments, the methods described herein are derived from cells that are distinct from the affinity-oligonucleotide conjugate or tetramer-oligonucleotide conjugate and different from a portion or copy thereof. And / or bar coding the polynucleotide in the vessel and / or the polynucleotide in the vessel. For example, a single cell encapsulated in a vessel that binds or binds to an affinity-oligonucleotide or tetramer-oligonucleotide conjugate can be lysed and is derived from a single cell Alternatively, additional polynucleotides, such as polynucleotides within a single cell, can be barcoded. In some embodiments, the oligonucleotide portion of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate in the vessel, e.g., the oligonucleotide portion that binds or is bound to a single cell in the vessel , And / or a copy or amplification product thereof can be barcoded using a Bessel barcode sequence. In some embodiments, one or more cellular polynucleotides from a single cell can be barcoded using the same Bessel barcoding sequence; such additional one or more cellular polynucleotides The nucleotides are further barcoded in some embodiments using a molecular barcode.

T cell receptor chain pairs and antibody immunoglobulin chain pairs are both types of immunoreceptors. In some embodiments, the cellular polynucleotide is an antibody immunoglobulin chain (or portion) encoding polynucleotide; in some embodiments, it is a TCR (or portion) encoding polynucleotide or Including. In some embodiments, the antigen bound by the affinity-oligonucleotide conjugate is a TCR or an antibody or a chain or portion thereof. In one aspect, the methods described herein further comprise generating a polynucleotide library for high-throughput sequencing and diagnostics. In one aspect, the methods described herein further comprise developing a human-derived library panel for antibody and / or TCR discovery from patients or cohorts with certain common characteristics. The disclosed invention combines multiple different types of paired variable sequences, such as T cell receptor chain pairs and antibody immunoglobulins, with the characterization of a single cell using an affinity-oligonucleotide conjugate. It can be applied to strand pairs. For example, a polynucleotide complementary to a cellular polynucleotide, eg, a heavy and / or light chain, eg, a _VH and / or _VL antibody chain and / or alpha and / or beta and / or gamma and / or Delta chains, eg, Vα / Vβ and Vγ / Vδ T cell receptor (TCR) chains (eg, from a framework portion thereof) can be introduced (or contained within the vessel) during formation of the vessel. obtain). A polynucleotide having a vessel barcode can also be introduced (or contained within the vessel) during formation of the vessel. These Bessel bar-coded polynucleotides are degenerated so that each cellular polynucleotide containing the Bessel bar code contains, during the reaction (s), a unique identity code corresponding to the vessel in which it resides. May hold a rate barcode. Thus, in some such embodiments, multiple polynucleotides having the same unique identity code are considered to originate from the same vessel, and thus, in some aspects, originate from a single cell. It is thought that there is. A plurality of polynucleotides having a molecular barcode can also be introduced during formation of the vessel, or be contained in the vessel. These molecular barcoding polynucleotides are degenerated barcodes such that each cellular polynucleotide molecule containing the molecular barcode contains a unique identity code corresponding to the single cellular polynucleotide molecule from which they are derived. Can be held. Millions of single immune cells can be lysed inside the emulsion and cell transcripts such as _VH and _VL and / or Vα / Vβ and / or Vγ / Vδ chain transcripts use primers Can be reverse transcribed or copied, then tagged with a vessel barcode and a molecular barcode, and the barcoded polynucleotide can be PCR amplified. Each _VH and _VL and / or Vα / Vβ and / or Vγ / Vδ chains that originate from a single immune cell (eg, a B cell or T cell) have virtually the same vessel barcode identity and Can be linked.

The _VH and _VL and / or Vα / Vβ and / or Vγ / Vδ chains can then be recovered from the vessel and PCR-enriched to add a next generation sequencing (NGS) tag. Libraries can be sequenced using a high-throughput sequencing platform, followed by repertoire diversity analysis, antibody frequency, CDR3 characterization, somatic hypermutation phylogenetic analysis, and the like. A database of precisely matched _VH and _VL and / or Vα / Vβ and / or Vγ / Vδ pairs can be generated by deconvolving Vessel and molecular barcode sequences. As each single immune cell is isolated in its respective vessel, for each vessel barcode observed twice, the sequenced transcripts originate from the same emulsion droplet and therefore have a unique Originated from a single cell. For sequences containing the same Bessel barcode, for each different molecular barcode observed, the sequenced transcripts originated from different transcript molecules from a single cell. For sequences containing the same vessel barcode, for each of the same molecular barcodes observed, the transcript sequenced originated from the same transcript molecule (eg, PCR overlap) from a single cell.

In parallel with the sequencing, libraries of _VH and _VL and / or Vα / Vβ and / or Vγ / Vδ chains recovered from the vessel can be cloned into an antibody expression vector and used for yeast display screening. It can be co-transfected. Cloning this same library pool is a preferred method compared to splitting a biological sample at the outset, since some rare immune cells may be used in one or the other assay. Because it is captured only at Libraries of _VH and _VL and / or Vα and Vβ and / or Vγ and Vδ chains from humans can be expressed regardless of the exact or incorrect pair matching, as in classical display assays. Yeast display can then be performed on one or more antigen targets to enrich for potential antibody candidates.

Positive candidate antibodies emerging from display technologies such as yeast display can be sequenced and queried against a matched paired barcode database. Each yeast-displayed _VH and / or Vα and / or Vγ chain can be rematched to its respective _VL or Vβ or Vδ chain, respectively, and each yeast-displayed _VL and / or Vβ and / or The Vδ chain can be rematched to its respective _VH or Vα or Vγ chain, respectively. These precisely matched candidates can be genes that are synthesized and expressed in mammalian cell lines and functionally validated against the target of interest. These candidates can be fully human antibodies and / or TCRs.

Samples In some embodiments, any sample containing a polynucleotide can be used in the methods described herein. In general, any sample containing cells can be used in the methods described herein. For example, the sample can be a biological sample from a subject, or a sample from a subject and containing RNA or DNA. The polynucleotide may be extracted from a biological sample, or the sample may be subjected directly to the method without extracting or purifying the polynucleotide. The sample can be extracted or isolated DNA or RNA. The sample may also be total RNA or DNA, a cDNA library, a virus, or genomic DNA extracted from a biological specimen. In one embodiment, the polynucleotide is isolated from a biological sample containing various other components such as proteins, lipids, and non-template nucleic acids. The nucleic acid template molecule can be obtained from any cellular material obtained from animals, plants, bacteria, fungi, or any other cellular organism. In certain embodiments, the polynucleotide is obtained from a single cell. Polynucleotides can be obtained directly from an organism or from a biological sample obtained from an organism. Any tissue or body fluid sample can be used as a source of the nucleic acids used in the present invention. Polynucleotides may also be isolated from cultured cells, such as primary cell cultures or cell lines. The cell or tissue from which the template nucleic acid is obtained may be infected with a virus or other intracellular pathogen.

  In certain embodiments, the antibodies or TCR-producing immune cells are immunized or infected, such as a human or other animal, such as a human or other animal with an infection, cancer, autoimmune condition, or any other disease. Pathogen-specific, tumor-specific, and / or disease-specific that may be isolated from the blood or other biological sample of a subject or host, such as a human or other animal, and may be of clinical significance Antibodies or TCRs can be identified. For example, a human may have been diagnosed with a disease and have shown symptoms of the disease, but may not have been diagnosed with the disease or may have no symptoms of the disease. For example, a human can be a human who has been exposed to an infectious agent (eg, a virus, bacterium, parasite, prion, etc.), an antigen, or a disease, and / or an infectious agent (eg, a virus, bacterium, parasite, prion, etc.), It may be an antigen, or a human capable of producing useful antibodies or TCRs against the disease. For example, the animal can be an animal that has been exposed to an infectious agent (eg, a virus, bacterium, parasite, prion, etc.), an antigen, or a disease, and / or an infectious agent (eg, a virus, bacterium, parasite, prion, etc.), It may be an animal capable of producing an antigen or a useful antibody or TCR against a disease. Certain immune cells from the immunized host make antibodies or TCRs against one or more target antigens and / or one or more unknown antigens of interest. In the present invention, the lymphocyte pool is subjected to fluorescence activated cell sorting (FACS), magnetically activated cell sorting (MACS) before the antibody chains are sequenced, antibodies are made, or an expression library is made. ) Using any suitable method, such as screening and sorting of cells using panning methods, or other screening methods for generating multiple immune cells from a sample, such as an immune cell library. Cells can be concentrated. In contrast to the prior art enrichment methods that provide only a small subset of immune cells expressing different antibodies, and thus only a small number of naturally occurring combinations of variable domains, the immune cell libraries of the present invention comprise: It contains at least two subsets of individual immune cells expressing different antibodies or TCRs. For example, the immune cell library of the present invention comprises at least 5, 10, 100, 250, 500, 750, 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000 of individual immune cells expressing different antibodies or TCRs. It may contain 10,000, 250,000, 500,000, 750,000, 1,000,000, 25,000,000, 5,000,000, 75,000,000, or 10,000,000 subsets. The method of the invention maximizes immune cell recovery and results in a very high degree of diversity.

T cells include peripheral blood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, or any other lymphoid tissue, and tumors. It can be obtained from several sources. T cells can be obtained from T cell lines and from autologous or allogeneic sources. The T cells may be obtained from a single individual or a population of individuals, for example, a population of individuals who all suffer from the same disease, such as cancer or infectious disease. In some embodiments, cells from the individual's circulating blood are obtained by apheresis or leukocyte apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis or leukocyte apheresis may be washed to remove the plasma fraction, and the cells may be placed in a buffer or medium suitable for subsequent processing. In one embodiment of the invention, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the cleaning solution may lack calcium, may lack magnesium, or may lack many, but not all, divalent cations. As those skilled in the art will immediately appreciate, the washing step can be accomplished by methods known to those skilled in the art, such as using a semi-automated "flow-through" centrifuge. After washing, the cells may be resuspended in various biocompatible buffers, such as, for example, Ca ++ / Mg ++ free PBS. Alternatively, undesired components of the apheresis sample may be removed and the cells may be resuspended directly in the culture medium. In other embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and centrifuging on a PERCOLL ™ gradient. Certain subpopulations of T cells, such as CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and CD45RO ⁺ T cells, may be further isolated by positive or negative selection techniques. For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (eg, DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In some embodiments, enrichment of the T cell population by negative selection can be achieved by combining antibodies against surface markers specific to the negatively selected cells. One such method is cell sorting and / or cell selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies to cell surface markers present on the negatively selected cells. For example, to enrich CD4 ⁺ cells by negative selection, monoclonal antibody cocktails typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Another method for preparing T cells for stimulation is to freeze the cells after a washing step, which does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and they will be useful in this context, one method is to use PBS containing 20% DMSO and 8% human serum albumin (HSA). Or using other suitable cell freezing media. It is then diluted 1: 1 with medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen at -80 ° C at 1 ° C per minute and stored in the gas phase in a liquid nitrogen storage tank.

In some embodiments, a non-immunized human donor or immune cells from a non-human donor are utilized. Animal naive repertoire (repertoire before antigen challenge) can be coupled to essentially any non-self molecule with moderate affinity (K _A of about ^{1 × 10 -6 ~1 × 10 -7} M) The antibody or TCR is provided to the animal. The sequence diversity of the antibody or TCR binding site is not directly encoded in the germline, but is assembled in a combinatorial fashion from V gene segments. Immunization, as described above, attached to the immunogen to proliferate (clonal expansion), corresponding to elicit any immune cells making V _H -V _L or V.alpha-V? Or V.gamma-V8 combined secrete antibodies I do. However, the use of spleen cells and / or immune cells or other peripheral blood lymphocytes (PBLs) from non-immunized subjects can provide a better presentation of possible antibodies or TCR repertoires. Also, subsequently, any animal species can be used to construct a B cell or T cell antibody or TCR library.

  In some cases, at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, A blood volume of 10, 20, 25, 30, 35, 40, 45, or 50 mL is collected.

In some cases, the starting material is peripheral blood. Peripheral blood cells can enrich for specific cell types (eg, monocytes, red blood cells, CD4 ⁺ cells, CD8 ⁺ cells, immune cells, T cells, or NK cells, etc.). In addition, peripheral blood cells can selectively deplete specific cell types (eg, mononuclear cells, red blood cells, CD4 ⁺ cells, CD8 ⁺ cells, immune cells, T cells, or NK cells, etc.).

  In some cases, the starting material can be a tissue sample containing solid tissue, including, but not limited to, brain, liver, lung, kidney, prostate, ovary, spleen, lymph nodes (such as tonsils). Thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus, and stomach. In other cases, the starting material may be cells containing nucleic acids, immune cells, and especially B cells or T cells. In some cases, the starting material may be a nucleic acid-containing sample from any organism from which genetic material can be obtained. In some cases, the sample is a fluid, for example, blood, saliva, lymph, or urine.

  A sample can be taken from a subject having a condition. In some cases, the subject from which the sample is taken may be a patient, eg, a cancer patient, or a patient suspected of having cancer. The subject can be a mammal, eg, a human, and can be male or female. In some cases, the female is pregnant. The sample may be a tumor biopsy. The biopsy is performed by a healthcare professional, including, for example, a physician, physician assistant, nurse, veterinarian, dentist, spine acupuncture, medical assistant, dermatologist, oncologist, gastroenterologist, or surgeon. Is also good.

  In some cases, non-nucleic acid material may be removed from the starting material using an enzymatic treatment (such as protease digestion).

  In some cases, blood is collected and stored at 4 ° C. in a device containing a magnesium chelator, including but not limited to EDTA. Optionally, a calcium chelator, including but not limited to EGTA, may be added. In other cases, but not limited to, formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, protein crosslinkers, nucleic acid crosslinkers, protein and nucleic acid crosslinkers, primary amine reactive crosslinkers, sulfhydryl reactions A cytolytic inhibitor is added to the blood, including a reactive crosslinker, sulfhydryl addition or disulfide reduction, a carbohydrate reactive crosslinker, a carboxyl reactive crosslinker, a photoreactive crosslinker, or a cleavable crosslinker.

  In some cases, where the extracted material comprises single-stranded RNA, double-stranded RNA, or DNA-RNA hybrids, these molecules are converted to double-stranded DNA using techniques known in the art. Can be converted. For example, DNA may be synthesized from RNA molecules using reverse transcriptase. In some cases, conversion of RNA to DNA may require a pre-ligation step to ligate the linker fragment to the RNA, and the use of universal primers allows the initiation of reverse transcription. In other cases, reverse transcription can be initiated, for example, using the polyA tail of the mRNA molecule. After conversion to DNA, in some cases, the desired sequences can be further captured, selected, tagged, or isolated using the methods detailed herein.

  Nucleic acid molecules include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). Nucleic acid molecules may be synthetic or derived from naturally occurring sources. In one embodiment, the nucleic acid molecule is isolated from a biological sample containing various other components such as proteins, lipids, and non-template nucleic acids. The nucleic acid template molecule can be obtained from any cellular material obtained from an animal, plant, bacterium, fungus, or any other cellular organism. In certain embodiments, the nucleic acid molecule is obtained from a single cell. Biological samples used in the present invention include virus particles or preparations. Nucleic acid molecules can be obtained directly from an organism or from biological samples obtained from an organism, such as blood, urine, cerebrospinal fluid, semen, saliva, sputum, feces, and tissues. Any tissue or body fluid sample can be used as a source of the nucleic acids used in the present invention. Nucleic acid molecules may also be isolated from cultured cells, such as primary cell cultures or cell lines. The cell or tissue from which the template nucleic acid is obtained may be infected with a virus or other intracellular pathogen.

  The sample may also be total RNA, a cDNA library, a virus, or genomic DNA extracted from a biological specimen. In certain embodiments, the nucleic acid molecule is conjugated to another target molecule, such as a protein, enzyme, substrate, antibody, binder, bead, small molecule, peptide, or any other molecule. In general, nucleic acids are extracted from biological samples by various techniques, such as those described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, NY (2001). be able to. The nucleic acid molecule can be single-stranded, double-stranded, or double-stranded (eg, a stem and loop structure) having a single-stranded region.

  DNA extraction methods are well known in the art. Classical DNA isolation protocols are based on extraction using an organic solvent, such as a mixture of phenol and chloroform, followed by precipitation with ethanol (J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989). , Second Edition, Cold Spring Harbor Laboratory Press: New York, NY). Other methods include the following: salting out DNA extraction method (P. Sunnucks et al., Genetics, 1996, 144: 747-756; SM Aljanabi et al., Nucl. Acids Res., 1997; 25: 4692-4693), trimethylammonium bromide salt DNA extraction method (S. Gustincich et al., BioTechniques, 1991, 11: 298-302), and guanidium thiocyanate DNA extraction method (JBW Hammond et al., Biochemistry). 1996, 240: 298-300). Various kits for extracting DNA from biological samples are commercially available (eg, BD Biosciences Clontech (Palo Alto, CA): Epicentre Technologies (Madison, Wis.); Gentra Systems, Inc. MicroProbe Corp. (Bothell, WA); Organon Teknika (Durham, NC); and Qiagen Inc. (Valencia, CA).

  RNA extraction methods are also well known in the art (eg, J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 211st edition, Cold Spring Harbor Laboratory Press: New York), and bodily fluids. Kits for extracting RNA from are commercially available (eg, Ambion, Inc. (Austin, TX); Amersham Biosciences (Piscataway, NJ); BD Biosciences Clontech (Palo Alto, CA); BioRad Laboratories (Hercules, Calif.); Dynal Biotech Inc. (Lake Success, NY); Epicenter Technologies (Madison, Wis.) Gentra Systems, Inc. (Minneapolis, MN); GIBCO BRL (Gaithersburg, MD); Invitrogen Life Technologies (Carlsbad, CA); MicroProbe Corp., Oklahoma, Oregon; Promega, Inc. (Madison, Wis.); And Qiagen Inc. (Valencia, CA).

  The one or more samples may be from one or more sources. One or more samples may be from two or more sources. The one or more samples may be from one or more subjects. The one or more samples may be from two or more subjects. The one or more samples may be from the same subject. The one or more subjects may be from the same species. The one or more subjects may be from different species. One or more subjects may be healthy. One or more subjects may be affected by the disease, disorder, or condition.

  In some embodiments, the sample is a fluid such as blood, saliva, lymph, urine, cerebrospinal fluid, semen, sputum, feces, or tissue homogenate.

  The sample may be taken from a subject having a condition. In some embodiments, the subject from which the sample is taken may be a patient, for example, a cancer patient, or a patient suspected of having cancer. The subject can be a mammal, eg, a human, and can be male or female. In some embodiments, the female is pregnant. The sample may be a tumor biopsy. The biopsy is performed by a healthcare professional, including, for example, a physician, physician assistant, nurse, veterinarian, dentist, spine acupuncture, medical assistant, dermatologist, oncologist, gastroenterologist, or surgeon. Is also good.

  In some embodiments, the polynucleotide is conjugated to another target molecule, such as a protein, enzyme, substrate, antibody, binder, bead, small molecule, peptide, or any other molecule. In some embodiments, the polynucleotide is not attached to a solid support. Nucleic acids can be extracted from biological samples by various techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, NY (2001)).

  In some embodiments, the sample is saliva. In some embodiments, the sample is whole blood. In some cases, to obtain a sufficient amount of polynucleotide for testing, at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, A blood volume of 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50 mL is collected. In some embodiments, blood can be collected in a device containing a magnesium chelator, including but not limited to EDTA, and stored at 4 ° C. Optionally, a calcium chelator, including but not limited to EGTA, may be added.

  In some embodiments, but not limited to, formaldehyde, formaldehyde derivatives, formalin, glutaraldehyde, glutaraldehyde derivatives, protein crosslinkers, nucleic acid crosslinkers, protein and nucleic acid crosslinkers, primary amine reactive crosslinkers, A cytolysis inhibitor, including a sulfhydryl-reactive crosslinker, sulfhydryl addition or disulfide reduction, a carbohydrate-reactive crosslinker, a carboxyl-reactive crosslinker, a photoreactive crosslinker, or a cleavable crosslinker, is added to blood. In some embodiments, non-nucleic acid material may be removed from the starting material using an enzymatic treatment (such as protease digestion).

  The plurality of samples may include at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, or more samples. The plurality of samples may include at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000, or more samples. The plurality of samples may be at least about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 samples, 9000 or 10,000 samples, or 100,000 samples, or 1,000,000. It may contain one or more samples. The plurality of samples may include at least about 10,000 samples.

  One or more polynucleotides in the first sample may be different from one or more polynucleotides in the second sample. One or more polynucleotides in the first sample may be different from one or more polynucleotides in the plurality of samples. One or more polynucleotides in the sample may include at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. . In some embodiments, the one or more polynucleotides in the sample comprises about 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than one nucleotide or base pair may be different. The plurality of polynucleotides in one or more samples of the plurality of samples may include two or more identical sequences. At least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% of the total polynucleotides in one or more of the plurality of samples %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% may contain the same sequence. The plurality of polynucleotides in one or more of the plurality of samples may include at least two different sequences. At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the total polynucleotides in one or more of the plurality of samples. %, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% may comprise at least two different sequences. In some embodiments, one or more polynucleotides are variants of each other. For example, one or more polynucleotides may contain genetic polymorphisms or other types of mutations. In another example, one or more of the polynucleotides is a splice variant.

  The first sample may include one or more cells, and the second sample may include one or more cells. One or more cells of the first sample may be of the same cell type as one or more cells of the second sample. One or more cells of the first sample may be of a different cell type than one or more different cells of the plurality of samples.

  Multiple samples may be obtained simultaneously. Multiple samples can be obtained simultaneously. A plurality of samples can be obtained sequentially. The plurality of samples can be obtained over an annual course of obtaining one or more different samples, for example, over 100, 10, 5, 4, 3, 2, or 1 year. . One or more samples can be obtained within about one year of obtaining one or more different samples. The one or more samples may be 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 4 months after obtaining one or more different samples Can be obtained within three, two, or one month. The one or more samples may be 30 days, 28 days, 26 days, 24 days, 21 days, 20 days, 18 days, 17 days, 16 days, 15 days, 14 days after obtaining one or more different samples. It can be obtained within days, 13, 12, 11, 10, 9, 8, 7, 6, 5, 5, 4, 3, 2, or 1 day. The one or more samples may be obtained at about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, after obtaining one or more different samples. It can be obtained within 4 hours, 2 hours, or 1 hour. The one or more samples can be obtained within about 60 seconds, 45 seconds, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second of obtaining one or more different samples. . The one or more samples can be obtained within less than 1 second of obtaining one or more different samples.

  Different polynucleotides of a sample may be present in the sample at different concentrations or amounts (eg, different numbers of molecules). For example, the concentration or amount of one polynucleotide may be greater than the concentration or amount of another polynucleotide in a sample. In some embodiments, the concentration or amount of at least one polynucleotide in the sample is at least about 1.5, 2, 3, 4, more than the concentration or amount of at least one other polynucleotide in the sample. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, It may be as large as 400, 500, 600, 700, 800, 900, 1000 times or more. In another example, the concentration or amount of one polynucleotide may be less than the concentration or amount of another polynucleotide in the sample. The concentration or amount of at least one polynucleotide in the sample is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, more than the concentration or amount of at least one other polynucleotide in the sample. , 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 , 800, 900, 1/1000, or greater.

  In some embodiments, two or more samples may contain different amounts or concentrations of polynucleotide. In some embodiments, the concentration or amount of one polynucleotide in one sample may be higher than the concentration or amount of the same polynucleotide in different samples. For example, a blood sample may contain a greater amount of a particular polynucleotide than a urine sample. Alternatively, a single sample can be split into two or more sub-samples. The sub-samples may contain different amounts or concentrations of the same polynucleotide. The concentration or amount of at least one polynucleotide in one sample is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, more than the concentration or amount of the same polynucleotide in another sample. , 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 , 800, 900, 1000 times, or more. Alternatively, the concentration or amount of one polynucleotide in one sample may be less than the concentration or amount of the same polynucleotide in a different sample. For example, the concentration or amount of at least one polynucleotide in one sample is at least about 1.5, 2, 3, 4, 5, 6, 7 more than the concentration or amount of the same polynucleotide in another sample. , 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600 , 700, 800, 900, 1/1000, or more.

Target Polynucleotides In some cases, the methods provided herein include target polynucleotide molecules such as polynucleotide molecules from cells, oligonucleotides of affinity-oligonucleotide conjugates, or their amplicons. For amplification and sequencing. In some cases, the methods provided herein relate to amplifying and sequencing at least one region of a target polynucleotide molecule. In some cases, the methods provided herein relate to amplifying and sequencing at least one target polynucleotide molecule. In one aspect, the target polynucleotide is an oligonucleotide of an affinity-oligonucleotide conjugate. In one aspect, the target polynucleotide is an RNA. In some embodiments, the target RNA polynucleotide is an mRNA. In some embodiments, the target RNA polynucleotide is polyadenylated. In some embodiments, the RNA polynucleotide is not polyadenylated. In some embodiments, the target polynucleotide is a DNA polynucleotide. For example, target polynucleotides include cDNA. The DNA polynucleotide may be genomic DNA. A DNA polynucleotide may include exons, introns, untranslated regions, or any combination thereof.

  In one aspect, the target polynucleotide is a genomic nucleic acid. DNA derived from genetic material in the chromosome of a particular organism may be genomic DNA. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of an antibody or TCR produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the heavy chain of an antibody produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the light chain of an antibody produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the alpha chain of a TCR produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the beta chain of a TCR produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the gamma chain of a TCR produced by an immune cell. In some embodiments, the target polynucleotide comprises a sequence comprising the variable region of the delta chain of a TCR produced by an immune cell. For example, the target polynucleotide may include the polynucleotide template used to generate the product of the reverse transcription or primer extension reaction, or may include the product of the reverse transcription or primer extension reaction itself. Good. For example, the target polynucleotide includes a target polynucleotide that can be subjected to a reverse transcription reaction or a primer extension reaction.

  In some embodiments, the target polynucleotide comprises a sequence comprising the AID of an oligonucleotide of an affinity-oligonucleotide or tetramer-oligonucleotide conjugate. In some embodiments, the target polynucleotide comprises a sequence comprising the AMD of the oligonucleotide of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate. For example, a target polynucleotide includes RNA or DNA. For example, target polynucleotides include synthetic oligonucleotides. For example, target polynucleotides include oligonucleotides containing AID and / or AMB.

The target polynucleotide can be obtained from virtually any source and can be prepared using methods known in the art. For example, the target polynucleotide may be prepared by methods known in the art, including, but not limited to, extracting genomic DNA or mRNA fragments from an organism or cell (eg, an immune cell) to obtain the target polynucleotide. Can be used and isolated directly without amplification. The target polynucleotide can also include cDNA generated from RNA (such as mRNA) by reverse transcription PCR. In some cases, the target polynucleotide is an RNA molecule. In some cases, the target polynucleotide is an mRNA molecule or a cDNA produced from the mRNA molecule. In some cases, the target polynucleotide is an mRNA molecule from a single immune cell or a cDNA molecule produced from the mRNA molecule. In some cases, the target polynucleotide is an mRNA molecule derived from individual immune cells or a cDNA molecule produced from those mRNA molecules. In some cases, the target polynucleotide is an mRNA molecule encoding an antibody sequence, derived from a single immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a heavy chain antibody sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a heavy chain antibody sequence, derived from a single immune cell. In some cases, the target polynucleotide is an mRNA molecule that encodes a light chain antibody sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a light chain antibody sequence, derived from a single immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding an antibody variable sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a variable antibody sequence, derived from a single immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a variable light chain antibody sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a variable light chain antibody sequence, derived from a single immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a variable heavy chain antibody sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a variable heavy chain antibody sequence from a single immune cell. In some cases, the target polynucleotide may be a cell-free nucleic acid, for example, DNA or RNA. In some cases, the target polynucleotide is an mRNA molecule encoding a variable alpha, beta, gamma, and / or delta chain TCR sequence from an individual immune cell. In some cases, the target polynucleotide is an mRNA molecule encoding a _VH and / or _VL BCR sequence from an individual immune cell.

  The methods described herein can be used to generate a library of polynucleotides for sequencing from one or more target polynucleotides. In some embodiments, a library may be generated from two or more regions of a target polynucleotide. In some embodiments, the method library may be generated from two or more target polynucleotides. In some embodiments, the target polynucleotide is a genomic nucleic acid or DNA from a chromosome. In some embodiments, the target polynucleotide comprises a sequence that includes a variant, such as a genetic polymorphism or mutation. In some embodiments, the target polynucleotide comprises DNA but not RNA. In some embodiments, the target polynucleotide comprises RNA but not DNA. In some embodiments, target polynucleotides include DNA and RNA. In some embodiments, the target polynucleotide is a single-stranded polynucleotide. In some embodiments, the target polynucleotide is a double-stranded polynucleotide. In some embodiments, the target polynucleotide is a single strand of a double-stranded polynucleotide.

  The target polynucleotide can be synthesized or obtained from any biological sample and can be prepared using methods known in the art. In some embodiments, the target polynucleotide is directly isolated without amplification. Methods for direct isolation are known in the art. Non-limiting examples include extracting genomic DNA or mRNA from a biological sample, organism, or cell. In some embodiments, one or more target polynucleotides are purified from a biological sample. In some embodiments, the target polynucleotide has not been purified from the biological sample containing it. In some embodiments, the target polynucleotide is isolated from a biological sample. In some embodiments, the target polynucleotide has not been isolated from the biological sample in which it is contained. In some embodiments, the target polynucleotide may be a cell-free nucleic acid. In some embodiments, the target polynucleotide may be a fragmented nucleic acid. In some embodiments, the target polynucleotide may be a transcribed nucleic acid. In some embodiments, the target polynucleotide is a modified polynucleotide. In some embodiments, the target polynucleotide is an unmodified polynucleotide.

  In some embodiments, the target polynucleotide is an oligonucleotide derived from an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate. In some embodiments, the plurality of target polynucleotides comprises a plurality of oligonucleotides from a plurality of affinity-oligonucleotide conjugates or a plurality of tetramer-oligonucleotide conjugates. In some embodiments, the plurality of target polynucleotides are derived from a plurality of affinity-oligonucleotide conjugates or tetramer-oligonucleotide conjugates, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, Include 850, 900, or 1000 or more oligonucleotides. In some embodiments, the plurality of target polynucleotides are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 or more affinity-oligonucleotide conjugates or Includes a plurality of oligonucleotides from the tetramer-oligonucleotide conjugate. In some embodiments, the plurality of target polynucleotides are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 or more affinity-oligonucleotide conjugates or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, derived from a tetramer-oligonucleotide conjugate 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 pieces or more Including the Kuno oligonucleotides.

In some embodiments, the target polynucleotide comprises an AID sequence. In some embodiments, the plurality of target polynucleotides are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, It includes 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 AID sequences or more. In some embodiments, the target polynucleotide comprises an AMB sequence. In some embodiments, the plurality of target polynucleotides are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500. , 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13 000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 , 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200 ^{000,300,000,400,000,500,000,600,000,700,000,800,000,900,000,1 × 10 6, 2 × 10} 6, 3 × 10 6, 4 × 10 6, 5 × 10 ⁶ , 6 × 10 ⁶ , 7 × 10 ⁶ , 8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ , 3 × 10 ⁷ , 4 × 10 ⁷ , 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ⁹ , 7 × 10 ⁹ , ^{8 × 10 9, 9 × 10} 9, 1 × 10 10, 2 × 10 10, 3 × 10 10, 4 × 10 10, 5 × 10 10, 6 × 10 10, ^{× 10 10, 8 × 10 10} , 9 × 10 10, 1 × 10 11, 2 × 10 11, 3 × 10 11, 4 × 10 11, 5 × 10 11, 6 × 10 11, 7 × 10 11, 8 × 10 ¹¹ , 9 × 10 ¹¹ , 1 × 10 ¹² , 2 × 10 ¹² , 3 × 10 ¹² , 4 × 10 ¹² , 5 × 10 ¹² , 6 × 10 ¹² , 7 × 10 ¹² , 8 × 10 ¹² , or Contains 9 × 10 ¹² or more AMB sequences.

  In some embodiments, the target polynucleotide is a polynucleotide from a single cell. In some embodiments, the target polynucleotide is from an individual cell. In some embodiments, the target polynucleotide is a polynucleotide from a sample containing a plurality of cells.

  In some embodiments, the target polynucleotide encodes a biomarker sequence. In some embodiments, the target polynucleotide encodes two or more biomarker sequences. In some embodiments, the plurality of target polynucleotides encode a biomarker sequence. In some embodiments, the plurality of target polynucleotides encode two or more biomarker sequences. In some embodiments, the plurality of target polynucleotides are 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, Or more biomarker sequences.

In some embodiments, the plurality of target polynucleotides comprises a panel of oligonucleotide sequences. In some embodiments, the plurality of target polynucleotides comprises a panel of immunoglobulin sequences. For example, a panel of immunoglobulin sequences may be _VH and / or _VL sequences. In some embodiments, the plurality of target polynucleotides comprises a panel of TCR sequences. In some embodiments, the panel of immunoglobulin or TCR sequences contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoglobulin or TCR sequences. In some embodiments, the plurality of target polynucleotides comprises a panel of BCR sequences. For example, a panel of BCR sequences may be _VH and / or _VL sequences. In some embodiments, the panel of immunoglobulin, BCR or TCR sequences has at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40, 000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, ^{00,000,200,000,300,000,400,000,500,000,600,000,700,000,800,000,900,000,1 × 10 6, 2 × 10} 6, 3 × 10 6 4 × 10 ⁶ , 5 × 10 ⁶ , 6 × 10 ⁶ , 7 × 10 ⁶ , 8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ , 3 × 10 ⁷ , 4 × 10 ⁷ 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ^{9 , 7 × 10 9, 8 ×} 10 9, 9 × 10 9, 1 × 10 10, 2 × 10 10, 3 × 10 10, 4 × 10 10, 5 × 10 ^{0, 6 × 10 10, 7} × 10 10, 8 × 10 10, 9 × 10 10, 1 × 10 11, 2 × 10 11, 3 × 10 11, 4 × 10 11, 5 × 10 11, 6 × 10 ¹¹ , 7 × 10 ¹¹ , 8 × 10 ¹¹ , 9 × 10 ¹¹ , 1 × 10 ¹² , 2 × 10 ¹² , 3 × 10 ¹² , 4 × 10 ¹² , 5 × 10 ¹² , 6 × 10 ¹² , 7 × 10 ^It contains ¹² , 8 × 10 ¹² , or 9 × 10 ¹² immunoglobulin, BCR or TCR sequences. In some embodiments, the panel of immunoglobulin, BCR or TCR sequences is at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350. , 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000. , 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40 000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1 ^{0,000,200,000,300,000,400,000,500,000,600,000,700,000,800,000,900,000,1 × 10 6, 2 × 10} 6, 3 × 10 6 4 × 10 ⁶ , 5 × 10 ⁶ , 6 × 10 ⁶ , 7 × 10 ⁶ , 8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ , 3 × 10 ⁷ , 4 × 10 ⁷ 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ^{9 , 7 × 10 9, 8 ×} 10 9, 9 × 10 9, 1 × 10 10, 2 × 10 10, 3 × 10 10, 4 × 10 10, 5 × 10 1 ^{, 6 × 10 10, 7 ×} 10 10, 8 × 10 10, 9 × 10 10, 1 × 10 11, 2 × 10 11, 3 × 10 11, 4 × 10 11, 5 × 10 11, 6 × 10 11 , 7 × 10 ¹¹ , 8 × 10 ¹¹ , 9 × 10 ¹¹ , 1 × 10 ¹² , 2 × 10 ¹² , 3 × 10 ¹² , 4 × 10 ¹² , 5 × 10 ¹² , 6 × 10 ¹² , 7 × 10 ¹² , 8 × 10 ¹² , or 9 × 10 ¹² immunoglobulin, BCR or TCR sequences. In some embodiments, the panel of immunoglobulin, BCR or TCR sequences is about 10-20, 10-30, 10-40, 10-30, 10-40, 10-50, 10-60, 10-70. , 10-80, 10-90, 10-100, 50-60, 50-70, 50-80, 50-90, 50-100, 100-200, 100-300, 100-400, 100-300, 100 -400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 500-600, 500-700, 500-800, 500-900, 500-1000, 1000-2000 , 1000-3000, 1000-4000, 1000-3000, 1000-4000, 1000-5000, 1000-6000 ^{1000~7000,1000~8000,1000~9000,1000~10000,5000~6000,5000~7000,5000~8000,5000~9000,5000~10000,1~1 × 10 5, 1~2 × 10} 5, 1-3 × 10 ⁵ , 1-4 × 10 ⁵ , 1-5 × 10 ⁵ , 1-6 × 10 ⁵ , 1-7 × 10 ⁵ , 1-8 × 10 ⁵ , 9 × 10 ⁵ , 1-1 × 10 ⁶ , 1-2 × 10 ⁶ , 1-3 × 10 ⁶ , 1-4 × 10 ⁶ , 1-5 × 10 ⁶ , 1-6 × 10 ⁶ , 1-7 × 10 ⁶ , 1-8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 1-2 × 10 ⁷ , 1-3 × 10 ⁷ , 1-4 × 10 ⁷ , 1-5 × 10 ⁷ , 1-6 × 10 ⁷ , 1-6 ^{7 × 10 7, 1~8 × 10} 7, 1~9 × 10 7, 1~1 × 10 8, 1~2 × 10 8, 1~3 × 1 ^{8, 1~4 × 10 8, 1~5} × 10 8, 1~6 × 10 8, 1~7 × 10 8, 1~8 × 10 8, 1~9 × 10 8, 1~1 × 10 9 , 1-2 × 10 ⁹ , 1-3 × 10 ⁹ , 1-4 × 10 ⁹ , 1-5 × 10 ⁹ , 1-6 × 10 ⁹ , 1-7 × 10 ⁹ , 1-8 × 10 ⁹ , 1-9 × 10 ⁹ , 1-1 × 10 ¹⁰ , 1-2 × 10 ¹⁰ , 1-3 × 10 ¹⁰ , 1-4 × 10 ¹⁰ , 1-5 × 10 ¹⁰ , 1-6 × 10 ¹⁰ , 1 ７7 × 10 ¹⁰ , 1 to 8 × 10 ¹⁰ , 1 to 9 × 10 ¹⁰ , 1 to 1 × 10 ¹¹ , 1 to 2 × 10 ¹¹ , 1 to 3 × 10 ¹¹ , 1 to 4 × 10 ¹¹ , 1 to 1 5 × 10 ¹¹ , 1-6 × 10 ¹¹ , 1-7 × 10 ¹¹ , 1-8 × 10 ¹¹ , 1-9 × 10 ¹¹ , 1-1 × 10 ¹² , 1-2 × 10 ¹² , 1-3 × ^{10 12,} 1~4 × ¹⁰ 12 ^{1~5 × 10 12, 1~6 × 10} 12, 1~7 × 10 12, 1~8 × 10 12 or 1 to 9 × ^{10 12} pieces of immunoglobulins, contain BCR or TCR sequences.

  In some embodiments, the target polynucleotide has a length of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450. , 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000 , 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 bases or base pairs. In some embodiments, the target polynucleotide has a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12, 000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 bases or base pairs. In some embodiments, the target polynucleotide is at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 in length. , 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12 000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 bases or base pairs. In some embodiments, the target polynucleotide has a length of about 10-20, 10-30, 10-40, 10-30, 10-40, 10-50, 10-60, 10-70, 10 -80, 10-90, 10-100, 50-60, 50-70, 50-80, 50-90, 50-100, 100-200, 100-300, 100-400, 100-300, 100-400 , 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 500-600, 500-700, 500-800, 500-900, 500-1000, 1000-2000, 1000 ~ 3000, 1000-4000, 1000-3000, 1000-4000, 1000-5000, 1000-6000, 1000-700 , 1000～8000,1000～9000,1000～10000,5000～6000,5000～7000,5000～8000,5000～9000, or 5,000 to 10,000 bases or base pairs. In some embodiments, the average length of the target polynucleotide or fragment thereof may be less than about 100, 200, 300, 400, 500, or 800 base pairs, or about 5, 10, 20, 20 , 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or less than 200 nucleotides, or about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, less than 100 kilobases. In some embodiments, the target sequence from a relatively short template, such as a sample containing the target polynucleotide, is about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases. In certain embodiments, the sequencing data is aligned to known or predicted sequences using a database containing sequences associated with a disease or condition or immunoglobulin or TCR sequences.

In some embodiments, the method further comprises determining a germline sequence of the first cellular polynucleotide, the second cellular polynucleotide, or both, wherein the first cellular polynucleotide is IgH or _VH. The second cellular polynucleotide comprises an IgL or _VL sequence, or any combination thereof. In some embodiments, the method comprises the sequence of IgL IgH, _VH , _VL , or any combination thereof from the sequence of germline IgL IgH, _VH , _VL , or any combination thereof. And determining the difference. In some embodiments, the method comprises: a total number of unique IgH sequences, a total number of unique IgL sequences; a total number of unique IgH and IgL sequences; a total number of unique paired IgL and IgH sequences; The method further comprises determining at least one of the frequency of the IgH or IgL sequence, or the frequency of the combination of the IgH and IgL sequences, relative to other sequences.

  In some embodiments, the method further comprises determining a germline sequence of the first cellular polynucleotide, the second cellular polynucleotide, or both, wherein the first cellular polynucleotide comprises a TCRa or Vα sequence. And the second cellular polynucleotide comprises a TCRβ or Vβ sequence, or any combination thereof. In some embodiments, the method comprises determining the sequence difference of TCRα, TCRβ, Vα, Vβ, or any combination thereof from the sequence of germline TCRα, TCRβ, Vα, Vβ, or any combination thereof. Further determining.

  In some embodiments, the method comprises: a total number of unique TCRa sequences, a total number of unique TCRa sequences; a total number of unique TCRa and TCRa sequences; a total number of unique matched TCRa and TCRa sequences; The method further comprises determining at least one of the frequency of the TCRa sequence or the TCRa sequence, or the frequency of the combination of the TCRa and TCRa sequences, relative to other sequences. In some embodiments, the method further comprises determining a germline sequence of the first cellular polynucleotide, the second cellular polynucleotide, or both, wherein the first cellular polynucleotide comprises a TCRγ or Vγ sequence. And the second cellular polynucleotide comprises a TCRδ or Vδ sequence, or any combination thereof. In some embodiments, the method comprises determining the sequence difference of the TCRγ, TCRδ, Vγ, Vδ, or any combination thereof from the sequence of the germline TCRγ, TCRδ, Vγ, Vδ, or any combination thereof. Further determining. In some embodiments, the method comprises: a total number of unique TCRγ sequences, a total number of unique TCRδ sequences; a total number of unique TCRγ and TCRδ sequences; a total number of unique paired TCRδ and TCRγ sequences; The method further includes determining at least one of the frequency of the TCRγ sequence or the TCRδ sequence, or the frequency of the combination of the TCRγ sequence and the TCRδ sequence, with respect to other sequences. In some embodiments, the method comprises: a total number of sequences from the first gene; a total number of sequences from the second gene; a total number of unique sequences from the first gene; Further comprising determining at least one of the total number of unique sequences derived from the gene; or the frequency of sequences derived from the first gene or sequences derived from the second gene.

In some embodiments, the method is based on individual paired IgL and IgH sequences, or one or more pairs of TCRα and TCRβ sequences, or TCRγ and TCRδ sequences, and the total amount of differences from the germline. , Antibodies or TCRs. In some embodiments, the method further comprises selecting an antibody or TCR based on one or more IgL or IgH sequences, TCRα and TCRβ sequences, or TCRγ and TCRδ sequences, and germline differences. . In some embodiments, the method further comprises selecting the antibody or TCR based on one or more of sequence pattern, analysis of differences, kinetics, or frequency. In some embodiments, the method further comprises selecting the antibody or TCR based on frequency.
Cloning and expression of antibodies, BCRs and TCRs

An “antibody expression library” or “BCR expression library” or “TCR expression library” or “expression library” as used herein is a collection of molecules (either at the nucleic acid or protein level). (I.e., two or more molecules). Thus, the term may refer to a collection of expression vectors (ie, at the nucleic acid level) encoding multiple antibodies, BCR or TCR molecules, or after they have been expressed in a suitable expression system (ie, (At the protein level), a collection of antibodies, BCR or TCR molecules. Alternatively, the expression vector / expression library may be contained in a suitable host cell capable of expressing them. The encoded or expressed antibody molecules in the expression libraries of the invention may be in any suitable format, for example, may be whole antibodies, BCR or TCR molecules, or antibodies, BCR fragments or TCRs. Fragments, for example, single chain antibodies (eg, scFv antibodies), Fv antibodies, Fab 'antibodies, (Fab') ₂ fragments, diabodies, and the like may be used. The terms "encoding" and "coding for", e.g., a nucleic acid sequence that "encodes" / "encodes" a particular enzyme, or a DNA coat sequence of a particular enzyme, or a particular enzyme. "Encoding" / "encoding" nucleotide sequence and the like, as well as other equivalent terms, refer to a DNA sequence that is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 'direction) coding sequence. A promoter is a part of a DNA sequence. This sequence region has an initiation codon at its 3 'end. The promoter sequence contains a minimal number of bases with the necessary elements to initiate transcription at a detectable level above background. However, when RNA polymerase binds to this sequence and transcription is initiated from the start codon (3 'end with promoter), transcription proceeds downstream in the 3' direction. Within the promoter sequence are found a transcription initiation site (conveniently defined by mapping with nuclease S1) as well as a protein binding domain (consensus sequence) that provides for the binding of RNA polymerase.

  Antibodies, BCR or TCR molecules identified by, derived from, selected from, or obtainable from an antibody, BCR or TCR expression library of the invention form a further aspect of the invention. Again, the antibody, BCR or TCR molecule can be a protein or nucleic acid encoding the antibody, BCR or TCR molecule, which can then be incorporated into a suitable expression vector, and / or May be contained in various host cells.

The cDNA pool is subjected to a PCR reaction with a polynucleotide that hybridizes to the constant region of the heavy chain of the antibody or BCR gene, and a polynucleotide that hybridizes to the 5 ′ end of the _VH or Vα or Vγ chain region of the antibody, BCR or TCR gene. May be served. The cDNA pool comprises the antibody, a polynucleotide that hybridizes to the heavy or constant region of the alpha or gamma chain of the BCR or TCR gene, and the _VH or Vα or Vγ chain of the antibody, BCR or TCR sequence that encodes a bar-coded polynucleotide comprising the sequence. The region may be subjected to a PCR reaction together with a polynucleotide that hybridizes to the 5 ′ to 5 ′ end of the region. PCR reactions can also be set up to amplify, for example, kappa and lambda class _VL or Vβ or Vδ chain pools. The cDNA pool is subjected to a PCR reaction with a polynucleotide that hybridizes to the constant region of the light chain of the antibody gene and a polynucleotide that hybridizes to the 5 ′ end of the _VL or Vβ or Vδ chain region of the antibody, BCR or TCR gene. You may. The cDNA pool comprises a polynucleotide that hybridizes to the constant region of the light chain of the antibody gene, and the region 5'-5 'of the _VL or Vβ or Vδ chain region of the antibody, barcoding polynucleotide comprising the BCR or TCR sequence. May be subjected to a PCR reaction together with a polynucleotide that hybridizes to the PCR. Such oligonucleotides or primers can be designed based on known and publicly available immunoglobulin, BCR or TCR gene sequence database information.

In some embodiments, the _VH and _VL or Vα and Vβ or Vγ and Vδ sequences are located in heavy or light chain genes, particularly the terminal regions of the _VH and _VL or Vα and Vβ or Vγ and Vδ polynucleotides. Can be conveniently obtained from a library of _VH and _VL or Vα and Vβ or Vγ and Vδ sequences produced by PCR amplification using one or more primers that are not specific for one or both of In some embodiments, the _VH and _VL sequences are _VH and _VL or Vα and Vα or Vβ or Vγ and Vδ produced by PCR amplification using primers specific for the region of the Vessel bar-encoding polynucleotide. It can be conveniently obtained from a library of sequences. In some embodiments, the _VH and _VL sequences are _VH and _VL or Vα and Vβ or Vγ and Vδ sequences produced by PCR amplification using C gene family-specific primers or C gene-specific primers. From the library. In some embodiments, the _VH and _VL sequences are a first primer specific to a region of the Bessel bar-encoding polynucleotide and a second primer that is a C gene family specific primer or a C gene specific primer. It can be conveniently obtained from a library of _VH and _VL or Vα and Vβ or Vγ and Vδ sequences produced by PCR amplification using a primer or a primer set having a plurality of second primers. In some embodiments, the _VH and _VL or Vα and Vβ or Vγ and Vδ sequences are a first primer specific to a region of the Bessel bar encoded polynucleotide and a second primer specific to a universal sequence. Can be conveniently obtained from a library of _VH and _VL or Vα and Vβ or Vγ and Vδ sequences produced by PCR amplification using a primer set having

In some embodiments, upon reverse transcription, the resulting cDNA sequence is converted to one of the terminal regions of a _VH and _VL or Vα and Vβ or Vγ and Vδ polynucleotides specific for an immunoglobulin or BCR gene. Alternatively, one or more primers specific for both may be used to amplify by PCR. In some embodiments, the _VH and _VL sequences are _VH and _VL or Vα and Vβ or Vγ and Vδ sequences produced by PCR amplification using V gene family-specific primers or V gene-specific primers. (Nicholls et al., J. Immunol. Meth., 1993, 165: 81; WO 93/12227) or are based on available information and are standard and known in the art. Designed according to the method. (The _VH and _VL or Vα and Vβ or Vγ and Vδ sequences are ligated, usually with an intervening spacer sequence (eg, encoding an in-frame flexible peptide spacer) to form a cassette encoding a single chain antibody. can do). The V region sequence can be conveniently cloned as a cDNA or PCR amplification product of an immunoglobulin expressing cell. The _VH and _VL or Vα and Vβ or Vγ and Vδ regions are optionally in the manner described herein, particularly after certain steps mentioned (eg, after single cell PCR). , After mammalian or other cell surface display, and after FACS screening). Sequencing may be used to confirm that the level of diversity is at an acceptable level, among other reasons. Sequencing may include high-throughput sequencing, deep sequencing (sequencing the same gene from multiple individual samples to identify sequence differences), or a combination of the two.

In some embodiments, using the methods described herein, it is not required that the natural _VH and _VL or Vα and Vβ or Vγ and Vδ combinations be physically linked. In some embodiments, the cDNA, barcoded polynucleotide, or PCR amplified barcoded cDNA is not physically linked. In some embodiments, the cDNA, barcoded polynucleotide, or PCR amplified barcoded cDNA is not physically linked in the same reaction or vessel.

In some embodiments, the native _VH and _VL or Vα and Vβ or Vγ and Vδ combination is one or more primers to the 5 ′ end of the _VH or Vα or Vγ gene in addition to the cDNA primer. And another primer or primers for the 5 'end of the _VL or Vβ or Vδ gene. These primers also contain a complementary tail, an additional sequence to allow self-assembly of the _VH and _VL or Vα and Vβ or Vγ and Vδ genes. Since the amplification and ligation reactions were performed in each cell, the likelihood of obtaining mixed products, in other words mixed variable regions, after PCR amplification and ligation is minimal. The risk of mixing is that bulky reagents such as digoxigenin-labeled nucleotides, to further assure that the V region cDNA pairs do not exit the cell compartment and intermix and remain inside the cell for PCR amplification and ligation. Can be further reduced by using. The amplified sequences are ligated by hybridization of complementary terminal sequences. After ligation, the sequence can be recovered from the cells used in the further method steps described herein. For example, the recovered DNA may be PCR amplified, optionally using terminal primers, and may be a plasmid, phage, cosmid, phagemid, viral vector, or a combination thereof, as detailed below. Can be cloned into a good vector. Convenient restriction sites may be incorporated into the hybridized sequence to facilitate cloning. Such vectors may also be stored as a library of linked variable regions for later use.

Expression systems can be selected to provide additional _VH and _VL or Vα and Vβ or Vγ and Vδ combinations. For example, bacteriophage expression systems allow random recombination of heavy and light chain sequences. Other suitable expression systems are known to those skilled in the art.

Note that for _VH and _VL or Vα and Vβ or Vγ and Vδ sequences from non-humans, in some embodiments it may be preferable to chimerize these sequences with fully human Fc. Should. As used herein, "chimerism" means that the heavy and light chain variable regions or Vα and Vβ or Vγ and Vδ regions are not of human origin and the heavy and light chains or Vα and Vβ or Vγ and Vδ are not human. Refers to an immunoglobulin, BCR or TCR in which the constant region of the chain is of human origin. This is achieved by amplifying and cloning the variable domain into human Fc. The human Fc can be part of a vector or in a separate molecule, and a library of Fc can be used. In a preferred embodiment, chimeric molecules grown in mammalian cells, such as CHO cells, are screened twice by FACS to enrich the cell population for cells expressing the antibody of interest. A chimeric antibody, BCR or TCR, is characterized either by sequencing and then functionally, or by direct functional characterization or kinetics. Growth, screening, and characterization are described in detail below.

  It is important to note that the PCR reaction described above has been described for cloning antibodies in the IgG form. IgG forms of antibody are preferred because they are generally associated with a more mature immune response, generally show higher affinity than IgM antibodies, and are more desirable for certain therapeutic or diagnostic applications. Obviously, however, if desired or appropriate, other forms of the immunoglobulin molecule, such as polynucleotides that will allow cloning of one or more of IgM, IgA, IgE, and IgD. Can be designed.

  If the integrity of the genetic material contained in the cells can be maintained, so that a library can be created at a later date, antibodies, BCRs or TCRs can be identified and the appropriate population of cells It is not necessary to generate an antibody, BCR or TCR expression library immediately after isolation at any time and optionally enrichment as described above. Thus, for example, the cells, cell lysates, or nucleic acids, eg, RNA or DNA derived therefrom, can be stored until a later date by any suitable method, eg, freezing, and the expression library can be stored at a later date when desired. Can be generated.

  Once a library of expression vectors has been generated, the encoded antibody molecules can be expressed in a suitable expression system and screened using suitable techniques that are well known and documented in the art. Accordingly, the method of the invention as defined above further comprises expressing the library of expression vectors in a suitable expression system, and screening the expressed library for antibodies having desired properties. It may be.

  As set forth herein, polynucleotides prepared by the methods of the present disclosure, including, but not limited to, polynucleotides encoding antibodies, BCRs or TCR sequences, include, but are not limited to, antibodies, BCRs or TCRs. Non-coding sequences of the entire amino acid sequence of the fragment, the antibody, BCR or TCR, or a portion thereof, the coding sequence of the antibody, BCR or TCR, fragment or portion, and having or having the additional coding sequence described above. Splicing and polyadenylation, such as at least one intron, together with additional non-coding sequences, including but not limited to non-coding 5 'and 3' sequences, such as the coding sequence of at least one signal leader or fusion peptide. Signal (eg, mRNA Additional sequences, such as transcribed, untranslated sequences that play a role in transcription, mRNA processing, including (somesome binding and stability); additional coding sequences that encode additional amino acids, such as those that provide additional functions. Is mentioned. Thus, the sequence encoding the antibody can be fused to a marker sequence, such as a fusion antibody comprising the antibody or TCR fragment or portion, or a sequence encoding a peptide that facilitates purification of the TCR.

Next, optionally, the primary PCR product is a new polynucleotide that hybridizes to the 5 ′ and 3 ′ ends of the antibody, BCR or TCR variable domains V _H , _VL kappa and _VL lambda or Vα and Vβ or Vγ and Vδ. The set may be subjected to a secondary PCR reaction (where appropriate, where a new set of polynucleotides is used, where the primary PCR reaction is a heavy or light chain antibody gene or a Vα or Vβ TCR gene or a portion of a Vγ or Vδ TCR gene). Depending on whether it was designed to amplify). These polynucleotides advantageously contain DNA sequences specific for a defined set of restriction enzymes (ie, restriction enzyme sites) for subsequent cloning. The restriction enzymes selected must be chosen so that cleavage does not occur within the human antibody, BCR or TCR V gene segments. Such polynucleotides can be designed based on known and publicly available immunoglobulin or TCR gene sequences and restriction enzyme database information. However, the preferred restriction enzyme sites included are NcoI, HindIII, MluI, and NotI. The product of such a secondary PCR reaction is a repertoire of various V-heavy, V-light chain kappa and V-light chain lambda antibody fragments / domains. Thus, if the expression library format of interest is the scFv or Fv format and only the _VH and _VL or Vα and Vβ or Vγ and Vδ domains of the antibody or TCR are present, this type of secondary A PCR reaction is performed.

The PCR product may also be subjected to a PCR reaction with a new set of primers that hybridizes to the 5 'and 3' ends of the barcoded polynucleotide. Advantageously, these polynucleotides can include DNA sequences specific for a defined set of restriction enzymes (ie, restriction enzyme sites) for subsequent cloning. The selected restriction enzyme must be chosen so that cleavage does not occur within the human antibody or TCR V gene segment. Such polynucleotides can be designed based on known and publicly available immunoglobulin, BCR or TCR gene sequences and restriction enzyme database information. However, the preferred restriction enzyme sites included are NcoI, HindIII, MluI, and NotI. The product of such a secondary PCR reaction is a repertoire of various _VH , _VL kappa, and _VL lambda antibody fragments / domains, or Vα and Vβ or Vγ and Vδ TCR fragments / domains.

  Heavy or light chains or Vα or Vβ chains or Vγ or Vδ chains Fv or Fab fragments, or single chain antibodies, BCRs or TCRs can also be used with this system. After mutating the heavy or light chain or the Vα or Vβ chain or the Vγ or Vδ chain, the complementary chain can be added to the solution. Thereafter, the two chains can be combined to form a functional antibody fragment. The addition of random, non-specific light or heavy chains or Vα or Vβ or Vγ or Vδ chain sequences allows for the production of combinatorial systems to generate libraries of diverse members.

  Antibodies derived from B or T lymphocytes of an immune-loaded host as defined herein, the variable heavy or Vα or Vγ chain region of the BCR or TCR gene or fragment thereof, and / or the variable light or Vβ chain or Such a repertoire library of cloned fragments, including the Vδ chain region or a fragment thereof, forms a further aspect of the invention. These libraries containing the cloned variable regions can optionally be inserted into an expression vector to form an expression library.

In some embodiments, the PCR reaction can be set up to retain all or part of the constant regions of the various antibodies, BCRs or TCR chains contained in the isolated immune cell population. This expression library format are the Fab format, heavy or alpha chain or gamma chain component comprises a V _H or Vα or Vγ and C _H or Cα or Cγ domain, light or Vβ chain or Vδ chain component but desirable when containing _{V L} or Vβ or Vδ chain and _{C L} or Cβ or Cδ domain. Again, a library of such cloned fragments containing all or part of the constant region of the antibody, BCR or TCR chain forms a further aspect of the invention.

  Such nucleic acids are convenient because they can include other sequences of the polynucleotide of the invention. For example, multiple cloning sites containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, translatable sequences can be inserted to assist in isolating the translated polynucleotide of the present invention. For example, a hexahistidine marker sequence provides a convenient means for purifying a protein of the invention. Optionally, the nucleic acid of the invention, excluding the coding sequence, is a vector, adapter, or linker for cloning and / or expressing a polynucleotide of the invention.

  Additional sequences may be added to such cloning and / or expression sequences to optimize their function during cloning and / or expression, to assist in isolating the polynucleotide, or to introduce the polynucleotide into cells. Can be improved. The use of cloning vectors, expression vectors, adapters, and linkers is well-known in the art. (See, eg, Ausubel, supra; or Sambrook, supra).

The libraries disclosed herein can be used for various applications. As used herein, a library contains a plurality of molecules. In some embodiments, the library comprises a plurality of polynucleotides. In some embodiments, the library comprises a plurality of primers. In some embodiments, the library includes multiple sequence reads from one or more polynucleotides, amplicons, or amplicon sets. The library can be stored and used multiple times to generate samples for analysis. Some uses include, for example, genotyping genetic polymorphisms, studying RNA processing, and selecting representative clones for sequencing by the methods provided herein. No. Libraries comprising a plurality of polynucleotides, such as primers or libraries for sequencing or amplification, can be generated, wherein the plurality of polynucleotides is at least about 2, 3, 4, 5, 6, 7, 8, , 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 , 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16 000, 17,000, 18,000, 19,000, 20,000, 30,000, 40,000, 50,000, 60 000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, Includes 1,000,000, 50,000,000, 100,000,000, or more molecular or Vessel barcodes. In some embodiments, the library of polynucleotides comprises a plurality of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 30 , 000,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 50,000,000, 100,000,000, or more It includes many unique polynucleotides, each unique polynucleotide including one or more molecular barcodes and Bessel barcodes.
barcode

  A molecular barcode, such as an antigenic molecule barcode, is a single molecule, e.g., a single oligonucleotide of an affinity-oligonucleotide or tetramer-oligonucleotide complex, or a single cell or single vessel. Contains information specific to polynucleotide molecules derived from Vessel barcodes are unique to polynucleotides from a single cell or present in a single vessel, as compared to polynucleotides from a different single cell or present in a different single vessel. Including information. In some embodiments, the unique information includes a unique sequence of nucleotides. For example, the sequence of a molecular or Bessel barcode can be determined by determining the identity and order of the unique or random sequence of nucleotides comprising the molecular or Bessel barcode. In some embodiments, unique information cannot be used to identify the sequence of the target polynucleotide. For example, a molecular barcode may be attached to one target polynucleotide, but a molecular barcode cannot be used to determine the attached target polynucleotide. In some embodiments, the unique information is not a known sequence linked to the identification of the target polynucleotide sequence. For example, a vessel barcode may be attached to one or more target polynucleotides, but the vessel barcode is used to determine any of the one or more target polynucleotides attached. It is not possible. In some embodiments, the unique information comprises a random sequence of nucleotides. In some embodiments, the unique information includes one or more unique sequences of nucleotides on the polynucleotide. In some embodiments, the unique information comprises a degenerate nucleotide sequence or a degenerate barcode. A degenerate barcode can include a variable nucleotide base composition or sequence. For example, the degenerate barcode may be in a random arrangement. In some embodiments, the complement sequence of the molecular or Bessel barcode is also a molecular or Bessel barcode sequence.

  Barcodes may include nucleotides of any length. For example, any of the barcodes described herein may have from 2 to 36 nucleotides, from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides. , From 2 to 20 nucleotides, from 4 to 20 nucleotides, or from 6 to 20 nucleotides. In certain aspects, the melting temperatures of the barcodes in the set are within 10 ° C of each other, within 5 ° C of each other, or within 2 ° C of each other. In certain aspects, the melting temperatures of the barcodes in the set are not within 10 ° C. of each other, within 5 ° C. of each other, or within 2 ° C. of each other. In some aspects, the barcode is a member of a set that has minimal cross-hybridization. For example, the nucleotide sequence of each member of such a set is such that the member cannot form a stable duplex with the complement of any other member under stringent hybridization conditions. May be sufficiently different from the nucleotide sequence of the other member. In some embodiments, the nucleotide sequence of each member of the set with minimal cross-hybridization differs from all other sequences by at least two nucleotides. Barcode technology is described in Winzeler et al. (1999) Science, 285: 901; Brenner (2000) Genome Biol., 1: 1 Kumar et al. (2001) Nature Rev., 2: 302; Giaever. Natl. Acad. Sci. USA, 101: 793; Eason et al. (2004) Proc. Natl. Acad. Sci. USA, 101: 11046; and Brenner (2004) Genome. Biol., 5: 240.

  For example, the barcode may include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, It may include 48, 49, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 nucleotides. For example, at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 60, 70, 80, 90, 100, 200, 500, or 1000 nucleotides. In some embodiments, the barcode has a specific length of nucleotides. For example, the barcode has a length of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 nucleotides.

  In some embodiments, each barcode in the plurality of barcodes has at least about 2 nucleotides. For example, each barcode in the plurality of barcodes has a length of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 nucleotides. In some embodiments, each barcode in the plurality of barcodes has at most about 1000 nucleotides. For example, each barcode in the plurality of barcodes has a length of at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, It may be 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 nucleotides.

  The number of molecular barcodes may be in excess of the total number of molecules to be labeled in multiple vessels. The number of Bessel barcodes may be in excess relative to the total number of molecules to be labeled in multiple vessels. For example, the number of molecular barcodes or Bessel barcodes is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 of the total number of molecules to be labeled in the plurality of vessels. , 30, 40, 50, 60, 70, 80, 90, or 100 times.

  The number of different molecular barcodes may be in excess of the total number of molecules to be labeled in multiple vessels. In some embodiments, the number of different molecular barcodes is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4 of the total number of molecules to be labeled in the plurality of vessels. , 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times.

  The number of different molecule barcodes in a single vessel may be in excess of the number of different molecules to be labeled in a single vessel. In some embodiments, the number of different molecular barcodes in a single vessel is at least about 1, 1.5, 2, 2.5, 3 of the number of different molecules to be labeled in a single vessel. , 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times.

  The number of different vessel barcodes may be less than the total number of molecules to be labeled in the plurality of vessels. In some embodiments, the number of different Bessel barcodes is at most about 1, 1.5, 2, 2.5, 3, 3.5, of the total number of molecules to be labeled in the plurality of vessels. 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 1/100.

  The number of amplification product molecules derived from a vessel bar-coded polynucleotide molecule in a single vessel may be in excess relative to the number of different molecules to be labeled in a single vessel. In some embodiments, the number of amplification product molecules from a Bessel bar-encoding polynucleotide molecule in a single vessel is at least about 1,1, .1 of the number of different molecules to be labeled in a single vessel. 5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, Or 100 times.

  The number of vessel bar-coded polynucleotide molecules in a single vessel may be less than the number of different molecules to be labeled in a single vessel. In some embodiments, the number of Bessel bar-coded polynucleotide molecules in a single vessel is at most about 1, 1.5, 2, 2 of the number of different molecules to be labeled in a single vessel. .5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 1 / 100th It is.

  The number of vessel bar-encoding polynucleotide molecules in a single vessel may be one molecule. The number of unamplified vessel bar-coded polynucleotide molecules in a single vessel may be one molecule.

  In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100 % Have the same concentration. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100 % Have the same concentration.

  In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100 % Have different concentrations. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100 % Have different concentrations.

The molecular barcode or Bessel barcode in the population of molecular barcodes or Bessel barcodes is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200. , 300, 400, 500, 600, 700, 800, 900, 1000, or more different arrangements. For example, molecular or Bessel barcodes in a population can have at least 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000. , 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100 000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or more different sequences May be provided. Accordingly, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 from one or more polynucleotides, such as a target polynucleotide, using multiple molecular or Bessel barcodes. , 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more different sequences can be generated. For example, using multiple molecular or Bessel barcodes, at least 2,000, 3,000, 4,000, 5,000, 6,000 from one or more polynucleotides, such as a target polynucleotide. , 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60 000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000 1 × 10 ⁶ , 2 × 10 ⁶ , 3 × 10 ⁶ , 4 × 10 ⁶ , 5 × 10 ⁶ , 6 × 10 ⁶ , 7 × 10 ⁶ , 8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ , 3 × 10 ⁷ , 4 × 10 ⁷ , 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ⁹ , 7 × 10 ⁹ , 8 × 10 ⁹ , 9 × 10 ⁹ , 1 × 10 ¹⁰ , 2 × 10 ¹⁰ , 3 × 10 ¹⁰ , 4 × 10 ¹⁰ , 5 × 10 ¹⁰ , 6 × 10 ¹⁰ , 7 × 10 ¹⁰ , 8 × 10 ¹⁰ , 9 × 10 ¹⁰ , 1 × 10 ¹¹ , 2 × 10 ¹¹ , 3 × 10 ¹¹ , 4 × 10 ^{11, 5 × 10 11, 6} × 10 11, 7 × 10 11, 8 × 10 11, 9 × 10 11, 1 × 10 12, 2 × 10 12, 3 × 10 12, 4 × 10 1 , It is possible to generate a ^{5 × 10 12, 6 × 10} 12, 7 × 10 12, 8 × 10 12, 9 × 10 12 atoms, or a number of different sequences than that. For example, using multiple molecular or Bessel barcodes, at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500, ^{000,600,000,700,000,800,000,900,000,1 × 10 6, 2 × 10} 6 ^{3 × 10 6, 4 × 10} 6, 5 × 10 6, 6 × 10 6, 7 × 10 6, 8 × 10 6, 9 × 10 6, 1 × 10 7, 2 × 10 7, 3 × 10 7, 4 × 10 ⁷ , 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ⁹ , 7 × 10 ⁹ , 8 × 10 ⁹ , 9 × 10 ⁹ , 1 × 10 ¹⁰ , 2 × 10 ¹⁰ , 3 × 10 ¹⁰ , 4 × 10 ¹⁰ , 5 × 10 ¹⁰ , 6 × 10 ¹⁰ , ^{7 × 10 10, 8 × 10} 10, 9 × 10 10, 1 × 10 11, 2 × 10 11, 3 × 10 11, 4 × 10 11, 5 × 10 11, 6 × 10 11, 7 × 10 11, ^{× 10 11, 9 × 10 11} , 1 × 10 12, 2 × 10 12, 3 × 10 12, 4 × 10 12, 5 × 10 12, 6 × 10 12, 7 × 10 12, 8 × 10 12, 9 X 10 ¹² or more target polynucleotides from at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200 ^{000,300,000,400,000,500,000,600,000,700,000,800,000,900,000,1 × 10 6, 2 × 10} 6, 3 × 10 6, 4 × 10 6, 5 × 10 ⁶ , 6 × 10 ⁶ , 7 × 10 ⁶ , 8 × 10 ⁶ , 9 × 10 ⁶ , 1 × 10 ⁷ , 2 × 10 ⁷ , 3 × 10 ⁷ , 4 × 10 ⁷ , 5 × 10 ⁷ , 6 × 10 ⁷ , 7 × 10 ⁷ , 8 × 10 ⁷ , 9 × 10 ⁷ , 1 × 10 ⁸ , 2 × 10 ⁸ , 3 × 10 ⁸ , 4 × 10 ⁸ , 5 × 10 ⁸ , 6 × 10 ⁸ , 7 × 10 ⁸ , 8 × 10 ⁸ , 9 × 10 ⁸ , 1 × 10 ⁹ , 2 × 10 ⁹ , 3 × 10 ⁹ , 4 × 10 ⁹ , 5 × 10 ⁹ , 6 × 10 ⁹ , 7 × 10 ⁹ , ^{8 × 10 9, 9 × 10} 9, 1 × 10 10, 2 × 10 10, 3 × 10 10, 4 × 10 10, 5 × 10 10, 6 × 10 10, ^{× 10 10, 8 × 10 10} , 9 × 10 10, 1 × 10 11, 2 × 10 11, 3 × 10 11, 4 × 10 11, 5 × 10 11, 6 × 10 11, 7 × 10 11, 8 × 10 ¹¹ , 9 × 10 ¹¹ , 1 × 10 ¹² , 2 × 10 ¹² , 3 × 10 ¹² , 4 × 10 ¹² , 5 × 10 ¹² , 6 × 10 ¹² , 7 × 10 ¹² , 8 × 10 ¹² , 9 × 10 ¹² or more different sequences can be generated.

  In some embodiments, sequences are grouped or binned using one or more molecular barcodes. In some embodiments, one or more molecular barcodes are used to group or sort the sequences into bins, with the sequence in each bin containing the same molecular barcode. In some embodiments, one or more molecular or Bessel barcodes are used to group or divide the sequences into bins, with the sequence in each bin comprising an amplicon set. In some embodiments, the sequence is grouped or binned using one or more molecular barcodes, wherein the sequence in each bin comprises a plurality of sequences, and wherein the plurality of sequences were generated Was derived from the same polynucleotide molecule in the amplification reaction.

  In some embodiments, one or more Bessel barcodes are used to group or bin arrays. In some embodiments, the array is grouped or binned using one or more Bessel barcodes, with the array in each bin containing the same Bessel barcode. In some embodiments, the array is grouped or binned using one or more Bessel barcodes, with the array in each bin including one or more amplicon sets. In some embodiments, the sequence is grouped or binned using one or more Bessel barcodes, wherein the sequence in each bin comprises a plurality of sequences, and wherein the plurality of sequences has been generated. Was derived from polynucleotide molecules from a single vessel or single cell.

  In some embodiments, one or more AID sequences are used to group or bin the sequences. In some embodiments, one or more AID sequences are used to group or sort the sequences, with the sequences in each bin containing the same AID sequence. In some embodiments, one or more AID sequences are used to group or divide the sequences into bins, with the sequence in each bin comprising one or more amplicon sets. In some embodiments, one or more AID sequences are used to group or divide the sequences into bins, wherein the sequence in each bin comprises a plurality of sequences, wherein the polynucleotide from which the plurality of sequences was generated is , A polynucleotide molecule derived from a single vessel or single cell.

  In some embodiments, one or more AMB sequences are used to group or bin the sequences. In some embodiments, one or more AMB sequences are used to group or divide the sequences into bins, with the sequences in each bin containing the same AMB sequence. In some embodiments, one or more AMB sequences are used to group or divide the sequences into bins, with the sequence in each bin comprising one or more amplicon sets. In some embodiments, one or more AMB sequences are used to group or divide the sequences into bins, wherein the sequence in each bin comprises a plurality of sequences, and wherein the polynucleotide from which the plurality of sequences was generated is , A polynucleotide molecule derived from a single vessel or single cell.

  In some embodiments, one or more AID and AMB sequences are used to group or bin the sequences. In some embodiments, one or more AID and AMB sequences are used to group or sort the sequences, with the sequences in each bin containing the same AID sequence. In some embodiments, one or more AID and AMB sequences are used to group or divide the sequences into bins, with the sequences in each bin containing the same AID sequence and different AMB sequences. In some embodiments, one or more AID and AMB sequences are used to group or divide the sequences into bins, with the sequences in each bin containing the same AID sequence and the same AMB sequence. In some embodiments, the sequences are grouped or binned using one or more AID and AMB sequences, with the sequence in each bin comprising one or more amplicon sets. In some embodiments, the sequences are grouped or binned using one or more AID and AMB sequences, wherein the sequence in each bin includes a plurality of sequences, and the plurality of sequences was generated. The polynucleotide was derived from the same polynucleotide in the amplification reaction, and was from a single cell or vessel.

  In some embodiments, one or more Bessel barcodes and AMB arrays are used to group or bin the arrays. In some embodiments, the array is grouped or binned using one or more Bessel barcodes and AMB arrays, with the array in each bin containing the same Bessel barcode. In some embodiments, one or more Bessel barcodes and AMB sequences are used to group or sort the sequences, with the sequences in each bin containing the same Bessel barcode and different AMB sequences. In some embodiments, one or more Bessel barcodes and AMB sequences are used to group or sort the sequences, with the sequences in each bin containing the same Bessel barcode and the same AMB sequence. In some embodiments, the array is grouped or binned using one or more Bessel barcodes and AMB arrays, with the array in each bin including one or more amplicon sets. In some embodiments, the array is grouped or binned using one or more Bessel barcodes and AMB arrays, wherein the array in each bin includes a plurality of arrays, and wherein the plurality of arrays is generated. The resulting polynucleotide was derived from the same polynucleotide in the amplification reaction and was derived from a single cell or vessel.

  In some embodiments, one or more AID arrays and Bessel barcodes are used to group or bin the arrays. In some embodiments, one or more AID sequences and Bessel barcodes are used to group or sort the sequences, with the sequences in each bin containing the same AID sequence. In some embodiments, one or more AID sequences and Bessel barcodes are used to group or sort the sequences into bins, with the sequences in each bin containing the same AID sequence and the same Bessel barcode. In some embodiments, one or more AID sequences and Bessel barcodes are used to group or sort the sequences into bins, with the sequence in each bin including one or more amplicon sets. In some embodiments, the arrays are grouped or binned using one or more AID arrays and Bessel barcodes, wherein the array in each bin includes a plurality of arrays, and wherein the plurality of arrays is generated. The resulting polynucleotide was derived from the same polynucleotide in the amplification reaction and was derived from a single cell or vessel.

  In some embodiments, sequences are grouped or binned using one or more molecular barcodes and Bessel barcodes. In some embodiments, the sequences are grouped or binned using one or more molecular barcodes and Bessel barcodes, wherein the sequences in each bin contain the same molecular barcode and the same Bessel barcode I do. In some embodiments, the sequences are grouped or binned using one or more molecular barcodes and Bessel barcodes, with the sequence in each bin comprising one or more amplicon sets. In some embodiments, one or more molecular barcodes and Bessel barcodes are used to group or divide the sequences into bins, wherein the sequence in each bin includes a plurality of sequences, and wherein the plurality of sequences is generated. The polynucleotides derived were from the same polynucleotide in the amplification reaction and were from a single cell or vessel. In some embodiments, the sequences are aligned without using one or more molecular barcodes and Bessel barcodes.

  In some embodiments, the sequences are aligned without using one or more molecular barcodes. In some embodiments, one or more molecular barcodes are used to align the sequences. In some embodiments, one or more molecular barcodes are used to group or sort the sequences and target-specific regions are used to align the sequences. In some embodiments, the arrays are aligned without using one or more Bessel barcodes. In some embodiments, the sequences are aligned using one or more Bessel barcodes. In some embodiments, one or more Bessel barcodes are used to group or sort the sequences and target-specific regions are used to align the sequences. In some embodiments, one or more molecular barcodes and Bessel barcodes are used to align the sequences. In some embodiments, one or more molecular barcodes and Bessel barcodes are used to group or bin the sequences and target-specific regions are used to align the sequences.

  In some embodiments, the aligned sequences contain the same AID. In some embodiments, the aligned sequences contain the same AMB. In some embodiments, the aligned sequences contain the same AID and AMB. In some embodiments, the aligned sequences contain the same AID and Bessel barcode. In some embodiments, the aligned sequences contain the same AMB and Bessel barcode. In some embodiments, the aligned sequences contain the same molecular barcode. In some embodiments, the aligned sequences contain the same Bessel barcode. In some embodiments, the aligned sequences contain the same molecular barcode and Bessel barcode. In some embodiments, the sequences are aligned using one or more molecular or Bessel barcodes, and the aligned sequences are two or more sequences from the amplicon set including. In some embodiments, one or more molecular or Vessel barcodes are used to align sequences, wherein the aligned sequences comprise a plurality of sequences, and wherein the plurality of sequences have been generated. Was derived from the same polynucleotide molecule in the amplification reaction. In some embodiments, one or more molecular or Vessel barcodes are used to align sequences, wherein the aligned sequences comprise a plurality of sequences, and wherein the plurality of sequences have been generated. Was from a single cell or a single vessel.

Droplet generation By dividing a sample of multiple cells into smaller reaction volumes and combining with multiple molecules of polynucleotides from individual cells or from individual cells and vessel barcoding, biomarkers High-throughput sequencing of a repertoire of sequences, such as sequences, may be possible.

  The transcriptome of an organism, by dividing a sample of multiple cells into small reaction volumes and combining with multiple molecules of polynucleotides from individual cells or from individual cells and Bessel barcoding High-throughput sequencing of a repertoire of sequences, such as sequences that represent a percentage of For example, the repertoire of sequences can be at least about 0.00001%, 0.00005%, 0.00010%, 0.00050%, 0.001%, 0.005%, 0.01% of the transcriptome of an organism. 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7% , 8%, 9%, 10%, 15%, 20%, 30%, 35%, 40%, 45, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 98%, 99%, or 100%.

  Splitting a sample of immune cells into small reaction volumes and combining heavy and light chains with multiple molecules of polynucleotides and polynucleotide molecules from individual immune cells or from individual cells and in conjunction with Bessel barcoding. High-throughput sequencing of a repertoire of strand sequences may be possible. Also, such methods may allow heavy and light chain pairing after sequencing based on the barcoding sequence. Also, dividing the sample into smaller reaction volumes as described herein may reduce reagent usage, thereby reducing material costs of the analysis.

  In some cases, the reverse transcription reaction and / or amplification reaction (eg, PCR) is performed in droplets, such as droplet digital PCR. In certain aspects, the invention provides a fluid compartment containing all or a portion of a target substance. In some embodiments, the compartment is a droplet. Although reference is made to "droplets" throughout the specification, the term is used interchangeably with fluid compartment and fluid distribution, unless otherwise specified. Unless otherwise specified, "droplets" are used for convenience and any fluid distribution or compartment can be used. Droplets as used herein include emulsion compositions (or mixtures of two or more immiscible fluids), such as those described in U.S. Patent No. 7,622,280. You may go out. Droplets can be generated by the device described in WO 2010/036352. The term "emulsion" as used herein may refer to a mixture of immiscible liquids (such as oil and water). The oil phase and / or the water-in-oil emulsion allows for compartmentalization of the reaction mixture within the aqueous droplets. Emulsions can include aqueous droplets within a continuous oil phase. The emulsion provided herein may be an oil-in-water emulsion where the droplets are oil droplets in a continuous aqueous phase. The droplets provided herein are designed to prevent inter-compartment mixing and each compartment protects its contents from evaporation and agglomeration with the contents of other compartments.

  The mixtures or emulsions described herein may be stable or unstable. The emulsion is relatively stable with minimal aggregation. Aggregation was said to have occurred when the small droplets combined to form progressively larger droplets. In some cases, 0.00001%, 0.00005%, 0.00010%, 0.00050%, 0.001%, 0.005%, 0.01% of the droplets generated from the droplet generator. %, 0.05%, 0.1%, 0.5%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, Less than 7%, 8%, 9%, or 10% aggregate with other droplets. Emulsions may also have limited flocculation, which is the process by which the dispersed phase precipitates out of suspension into flakes.

  About 0.001, 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180 , 200, 300, 400, or 500 microns, or about 0.001, 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, Less than 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or 500 microns, or about 0.001, 0.01, 0.05, 0.1, 1, 5, 10, Greater than 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or 500 microns, or at least about 0.001. 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, Droplets having an average diameter of 400 or 500 microns can be produced. The droplets can be from about 0.001 to about 500, about 0.01 to about 500, about 0.1 to about 500, about 0.1 to about 100, about 0.01 to about 100, or about 1 to about 100. It may have an average diameter of microns. Microfluidic methods for producing emulsion droplets are known in which either monodisperse or polydisperse emulsions are produced using microchannel cross-flow focusing or physical agitation. is there. The droplet may be a monodisperse droplet. Droplets can be generated such that the size of the droplet does not vary more than plus or minus 5% of the average size of the droplet. In some cases, the droplets are generated such that the droplet size does not vary by more than plus or minus 2% of the average droplet size. Droplet generators can produce a population of droplets from a single sample, where each droplet is plus or minus about 0.1%, 0.5%, or about the average size of the entire population of droplets. 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7% , 7.5%, 8%, 8.5%, 9%, 9.5%, or more than 10% in size.

  Higher mechanical stability may be useful for microfluidic manipulation and fluid handling at higher shear (eg, by microfluidic capillaries or by 90 degree bends in valves or the like in the fluid path). Droplets or capsules before or after heat treatment can be mechanically stable to standard pipetting and centrifugation.

  Droplets can be formed by flowing an oil phase through an aqueous sample. The aqueous phase comprises cells, nucleotides, nucleotide analogs, molecular barcoded polynucleotides, vessel barcoded polynucleotides, primers, template nucleic acids, and enzymes such as DNA polymerase, RNA polymerase, and / or reverse transcriptase. It may include a buffer solution and a reagent for performing an amplification reaction.

The aqueous phase may contain buffer solutions and reagents for performing the amplification reaction with or without solid surfaces such as beads. The buffer solution may be about 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM, more than about 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM, or It may contain less than about 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM Tris. In some cases, the concentration of potassium chloride is about 10, 20, 30, 40, 50, 60, 80, 100, 200 mM or about 10, 20, 30, 40, 50, 60, 80, 100. , 200 mM, or less than about 10, 20, 30, 40, 50, 60, 80, 100, 200 mM. The buffer solution may contain about 15 mM Tris and 50 mM KCl. The nucleotides are at a concentration greater than about 50, 100, 200, 300, 400, 500, 600, or 700 μm, or about 50, 100, 200, 300, 400, 500, 600, or 700 μm, respectively, or about It may comprise a deoxyribonucleotide triphosphate molecule comprising dATP, dCTP, dGTP, and dTTP of less than 50, 100, 200, 300, 400, 500, 600, or 700 μm. In some cases, the dUTP is about 50, 100, 200, 300, 400, 500, 600, or 700, 800, 900, or 1000 μm, about 50, 100, 200, 300, 400, 500, 600, Or added into the aqueous phase to a concentration greater than 700, 800, 900, or 1000 μm, or less than about 50, 100, 200, 300, 400, 500, 600, or 700, 800, 900, or 1000 μm. Is done. In some cases, magnesium chloride or magnesium acetate (MgCl ₂ ) comprises about 1.0, 2.0, 3.0, 4.0, or 5.0 mM of about 1.0, 2.0, 3 It is added to the aqueous phase at a concentration greater than 0.0, 4.0, or 5.0 mM, or less than about 1.0, 2.0, 3.0, 4.0, or 5.0 mM. The concentration of MgCl ₂ may be about 3.2 mM. In some cases, magnesium acetate or magnesium is used. In some cases, magnesium sulfate is used.

  Non-specific blocking agents such as BSA or gelatin from bovine skin can be used, with gelatin or BSA being present in a concentration range of approximately 0.1-0.9% w / v. Other possible blocking agents can include beta-lactoglobulin, casein, milk powder, or other common blocking agents. In some cases, the preferred concentration of BSA and gelatin is about 0.1% w / v.

  The amplification primers in the aqueous phase were about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, or 2.0 μm, about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7 or 2.0 μm or about 0.05, 0.1, 0.2 , 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, or less than 2.0 μm May be provided. The primer concentration in the aqueous phase can be from about 0.05 to about 2, about 0.1 to about 1.0, about 0.2 to about 1.0, about 0.3 to about 1.0, about 0.4 To about 1.0, or about 0.5 to about 1.0 μm. The concentration of the primer may be about 0.5 μm. The allowable range of the target nucleic acid concentration in PCR is not limited, but includes between about 1 pg and about 500 ng.

  Also, in some cases, the aqueous phase may include additives, including, but not limited to, non-specific background / blocking nucleic acids (eg, salmon sperm DNA), biopreser Biopreservatives (eg, sodium azide), PCR enhancers (eg, betaine, trehalose, etc.), and inhibitors (eg, RNAse inhibitors). As other additives, for example, dimethyl sulfoxide (DMSO), glycerol, betaine (mono) hydrate (N, N, N-trimethylglycine = [carboxy-methyl] trimethylammonium, trehalose, 7-deaza) -2'-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2'-dGTP), BSA (bovine serum albumin), formamide (methaneamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (eg, , Tetraethyammonium chloride (TEA-Cl) and tetrapropylammonium chloride (TPrA-Cl), non-ionic detergents (eg, Triton X-100, Tween 20, Nonidet P-40). NP-40)), or PREXCEL-Q. In some cases, the aqueous phase comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 species. In other cases, the aqueous phase may include at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different additives. It may be.

  In some cases, the non-ionic ethylene oxide / propylene oxide block copolymer is reduced to about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0. It may be added to the aqueous phase at a concentration of 7%, 0.8%, 0.9%, or 1.0%. Common biosurfactants include nonionic surfactants such as Pluronic F-68, Tetronics, and Zonyl FSN. Pluronic F-68 may be present at a concentration of about 0.5% w / v.

  In some cases, magnesium sulfate can be used at similar concentrations instead of magnesium chloride. A wide range of common PCR buffers available from various suppliers can be used in place of the buffer solution.

  Emulsions can be formulated to produce highly monodisperse droplets with a liquid-like interfacial film that can be converted into microcapsules with a solid-like interfacial film by heating. Such microcapsules can act as a bioreactor capable of retaining the contents during a reaction process such as PCR amplification. Conversion to microcapsule form may occur upon heating. For example, such conversion may occur at a temperature greater than about 50 ° C, 60 ° C, 70 ° C, 80 ° C, 90 ° C, or 95 ° C. In some cases, this heating occurs using a thermocycler. During the heating process, a fluid or mineral oil overlay may be used to prevent evaporation. Excess continuous phase oil may or may not be removed before heating. Biocompatible capsules may be resistant to flocculation and / or flocculation with a wide range of thermal and mechanical treatments. After conversion, the capsules can be heated at about 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, or 40 ° C. At a temperature higher than about 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, or 40 ° C; Alternatively, it can be stored at less than about 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, or 40 ° C. it can. Such capsules are for biomedical applications, such as stable digitized encapsulation of aqueous biological fluids containing macromolecules, especially nucleic acids or proteins or a mixture of both; drug and vaccine delivery; biomolecular libraries; and clinical imaging applications May be useful for strategic applications.

  The microcapsules may contain one or more polynucleotides and may be resistant to aggregation, especially at elevated temperatures. Thus, PCR amplification reactions may occur at very high densities (eg, the number of reactions per unit volume). In some cases, 100,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 5,000,000, or 10,000 per ml More than 2,000 separate reactions may occur. In some cases, the reaction occurs in a single well, eg, in a well of a microtiter plate, without causing intermixing between the reaction volumes. The microcapsules may also contain other components necessary to allow the reverse transcription, primer extension, and / or PCR reaction to occur, such as primers, probes, dNTPs, DNA or RNA polymerase, etc. Good. Such capsules are resistant to flocculation and flocculation with a wide range of thermal and mechanical treatments.

  In some cases, the amplification step is performed by performing a digital PCR, such as a microfluidic-based digital PCR or a droplet digital PCR.

  Droplets can be generated using a microfluidic system or device. As used herein, the prefix “micro” (eg, for “microchannel” or “microfluid”) is generally less than about 1 mm, and in some cases, about 100 microns ( (Micrometer). In some cases, the element or article includes a channel through which fluid can flow. In addition, “microfluid” as used herein refers to a device, apparatus, or system that includes at least one microscale channel.

  Microfluidic systems and devices have been described in various contexts, typically in the context of small laboratory (eg, clinical) analysis. Other uses have been described as well. For example, International Patent Application Publication Nos. 063227.

  The droplet generally contains an amount of the first sample fluid in the second carrier fluid. Any technique for forming droplets known in the art can be used with the method of the present invention. An exemplary method includes flowing a flow of a sample fluid containing a target substance (eg, an immune cell) to intersect two opposing flows of a flowing carrier fluid. The carrier fluid is immiscible with the sample fluid. Intersecting the sample fluid with the two opposing flows of the flowing carrier fluid results in distribution of the sample fluid into individual sample droplets containing the target substance.

  The carrier fluid may be any fluid that is immiscible with the sample fluid. An exemplary carrier fluid is oil. In certain embodiments, the carrier fluid includes a surfactant.

  The same method is applied to contain other reagents, such as reagents for non-PCR based amplification reactions such as polymerase chain reaction (PCR), multiple strand displacement amplification, or other methods known to those skilled in the art. Individual droplets can be created. Suitable reagents for performing a PCR-based amplification reaction are known to those of skill in the art and include, but are not limited to, DNA polymerase, forward and reverse primers, deoxyribonucleotide triphosphate (dNTP), and one or more buffers. Is mentioned.

  In certain embodiments, the fluid compartment comprises a first fluid distribution (eg, droplets) containing a target substance (eg, a solid support such as immune cells and / or beads) and a second fluid (eg, a fluid). As a stream or within a droplet). The first and second fluids join to form a droplet. Merging may be achieved by applying an electric field to the two fluids. In certain embodiments, the second fluid contains reagents for performing an amplification reaction, such as a polymerase chain reaction or an amplification reaction.

  In certain aspects, the present invention relates to generating a library of uniquely barcoded heavy and light chain antibody sequences and / or alpha and beta chain TCR sequences and / or gamma and delta chain TCR sequences. A method is provided wherein each construct comprises obtaining a plurality of nucleic acid constructs comprising a unique N-mer and a functional N-mer. The functional N-mer may be a random N-mer, a PCR primer, a universal primer, an antibody, a sticky end, or any other sequence. The method may include creating M sets of N fluid compartments, each containing one or more copies of the unique construct. The method described above is more advanced by adding additional constructs to each compartment in the set and repeating this for each set to generate N × M compartments, each containing a unique pair of constructs. It is possible to create a barcode library having complicated complexity. These pairs may be hybridized or ligated to generate new constructs. For each construct in the barcode library, each unique N-mer may be suitable for identification by sequencing, probe hybridization, other methods, or a combination of methods.

Droplet Library In general, a drop library consists of several library elements that are pooled together in a single collection. Libraries can vary in complexity from a single library element to 1 × 10 ¹⁵ or more library elements. Each library element is a fixed concentration of one or more given components. Elements can be, but are not limited to, cells, beads, amino acids, proteins, polypeptides, nucleic acids, polynucleotides, or small molecule compounds. The element may contain an identifier, such as a molecular barcode, a vessel barcode, or both.

  Cell library elements can include, but are not limited to, hybridomas, B cells, T cells, primary cells, cultured cell lines, cancer cells, stem cells, or any other cell type. Cell library elements are prepared by encapsulating one to tens of thousands of cells in individual droplets. The number of encapsulated cells is usually determined by Poisson statistics from cell number density and droplet volume. However, in some cases, the numbers deviate from Poisson statistics as described in Edd et al., Lab Chip, 8 (8): 1262-1264, 2008. Because the cells are dispersive in nature, preparing the library into a population having multiple cell variants, such as immune cells each producing one antibody or TCR, all present in a single starting medium It is then possible to divide the medium into individual droplet capsules containing at most one cell. The cells in the individual droplet capsules are then lysed and the heavy and light chain polynucleotides and / or alpha and beta chain polynucleotides and / or gamma and delta chain polynucleotides derived from the lysed cells are converted to molecular barcodes and Bar-coded in a vessel, amplified and then combined or pooled to form a library composed of heavy and light chains and / or alpha and beta chains and / or gamma and delta chain library elements.

  Bead-based library elements contain one or more beads and may contain other reagents, such as antibodies, enzymes, or other proteins. If all library elements contain different types of beads, but the surrounding medium is the same, the library elements may all be prepared from a single starting fluid, or have different starting fluids. May be. For a library of cells prepared into a population from a collection of variants, the library elements will be prepared from various starting fluids. When starting with multiple cells, it is desirable to have exactly one cell per droplet and very few droplets containing more than one cell. In some cases, the number of drops containing exactly one cell per drop will be higher and the number of empty drops or drops containing more than one cell will be fewer exceptions. , Variations from Poisson statistics can be realized to provide enhanced droplet loading.

  In some embodiments, starting with more than one Vessel barcoding polynucleotide, having exactly one Vessel barcoding polynucleotide per droplet and more than one Vessel barcoding polynucleotide It is desirable that the number of droplets containing the polynucleotide be very small. In some cases, more droplets will have exactly one vessel bar-encoding polynucleotide per droplet, empty droplets or droplets containing more than one vessel bar-encoding polynucleotide Variations from Poisson statistics can be realized to provide enhanced drop-loading, such that is a small exception.

  Examples of droplet libraries are collections of droplets having a variety of different contents, such as beads, cells, small molecules, DNA, primers, antibodies, and bar-coded polynucleotides. Droplet sizes range in diameter from approximately 0.5 micron to 500 microns, which corresponds to about 1 picoliter to 1 nanoliter. However, the droplets can be as small as 5 microns or as large as 500 microns. Preferably, the droplets are less than 100 microns in diameter, from about 1 micron to about 100 microns. The most preferred size is about 20-40 microns (10-100 picoliters) in diameter. Preferred properties considered of the droplet library include osmotic balance, uniform size, and size range.

  The droplets contained within the droplet library provided by the present invention are preferably uniform in size. That is, the diameter of any droplet in the library is 5%, 4%, 3%, 2%, 1%, or 0.5% compared to the diameter of other droplets in the same library. Will vary less than. Uniform size of the droplets in the library can be important for maintaining the stability and integrity of the droplets, and will later be used in various biological assays described herein. And for chemical assays, it may be essential to use the droplets in the library.

  The present invention provides a droplet library comprising a plurality of aqueous droplets in an immiscible fluid, each droplet preferably being substantially uniform in size and including different library elements. The present invention is a method for forming a droplet library, comprising providing a single aqueous fluid containing different library elements, placing each library element in an aqueous droplet in an immiscible fluid. A method is provided that includes encapsulating.

  In certain embodiments, different types of elements (eg, cells or beads) are pooled into a single source contained in the same medium. After performing the initial pool, the elements are encapsulated in droplets to create a library of droplets, each droplet having a different type of bead or cell being a different library element. Diluting the initial solution enables the encapsulation process. In some embodiments, the droplets that are formed will either contain a single element or will contain no, ie, empty. In other embodiments, the droplets formed will contain multiple copies of the library element. The encapsulated element is generally a type of variant. In one example, the elements are immune cells of a blood sample, each immune cell being encapsulated to amplify and barcode the nucleotide sequence of an antibody within the immune cells.

  For example, in one type of emulsion library, there are library elements that have different particles, ie, cells or barcoding polynucleotides in different media, and are encapsulated before pooling. In one example, a specified number of library elements, i.e., n different cells or barcoded polynucleotides, are contained in different media. Each of the library elements is separately emulsified and pooled, at which point each of the n pooled different library elements is combined and pooled into a single pool. The resulting pool contains a plurality of water-in-oil emulsion droplets, each containing a different type of particle.

  In some embodiments, the droplets formed will either contain a single library element or will contain no, ie, empty. In other embodiments, the droplets formed will contain multiple copies of the library element. The content of the beads follows a Poisson distribution, where events occur at a known average rate and are temporally independent of the immediately preceding event, with a discrete probability distribution representing the probability of several events occurring over a period of time. Exists. The oils and surfactants used to create the library prevent the contents of the library from being exchanged between droplets.

Primers In general, one or more pairs of primers can be used in an amplification reaction. One primer of the primer pair may be a forward primer, and one primer of the primer pair may be a reverse primer.

In some cases, a first pair of primers can be used in an amplification reaction. One primer of the first pair may be a forward primer complementary to the sequence of the first target polynucleotide molecule, one primer of the first pair may be a reverse primer, It may be complementary to a second sequence of one target polynucleotide molecule, and the first target locus may be between the first sequence and the second sequence. In some embodiments, the first target locus comprises a _VH or Vα or Vγ sequence. In some embodiments, the first target locus comprises an AID sequence and / or an AMB sequence.

In some cases, a second pair of primers can be used in the amplification reaction. One primer of the second pair may be a forward primer complementary to the first sequence of the second target polynucleotide molecule, and one primer of the second pair may be a second target polynucleotide. The second primer may be a reverse primer that is complementary to the second sequence of the molecule, and the second target locus may be between the first and second sequences. In some embodiments, the second target locus comprises a _VL or Vβ or Vδ sequence.

  In some cases, a third pair of primers can be used in the amplification reaction. The third pair of one primer may be a forward primer complementary to the first sequence of the third target polynucleotide molecule, and the third pair of one primer may be a third target polynucleotide. The third primer may be a reverse primer complementary to the second sequence of the molecule, and the third target locus may be located between the first and second sequences. In some embodiments, the third target locus comprises a barcode, such as a molecular or Vessel barcode.

The length of the forward and reverse primers may depend on the sequence of the target polynucleotide and target locus. For example, the length and / or T _M of the forward and reverse primers can be optimized. In some cases, the primer has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or More than 60 or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, or less than 60 nucleotides. In some cases, the primer has a length of about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 15 to about 45, 15 to about 55, about 15 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, about 20 to About 55, about 20 to about 60 nucleotides.

  The primer may be single-stranded DNA before binding to the template polynucleotide. In some cases, the primer initially contains a double-stranded sequence. The appropriate length of a primer may depend on the intended use of the primer, but may range from about 6 to about 50 nucleotides, or from about 15 to about 35 nucleotides. For short primer molecules, lower temperatures may generally be required to form a sufficiently stable hybrid complex with the template. In some embodiments, the primer need not reflect the exact sequence of the template nucleic acid, but only need to be sufficiently complementary to hybridize with the template. In some cases, the primer may be partially double-stranded before binding to the template polynucleotide. Primers having a double-stranded sequence can have about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 primers. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more, or about 3, 4, 5, It may have a hairpin loop of less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases. The double-stranded portion of the primer is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, or 50 of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, or more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,. 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or less than 50 or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 base pairs. The design of suitable primers for amplifying a given target sequence is well known in the art.

  Primers can incorporate additional features that allow the detection or immobilization of the primer, but do not alter the basic properties of the primer (eg, acting as a starting point for DNA synthesis). For example, the primers may not hybridize to the target nucleic acid, but contain additional nucleic acid sequences at the 5 'end that facilitate cloning or further amplification or sequencing of the amplification product. For example, the additional sequence may include a primer binding site, such as a universal primer binding site. The region of the primer that has sufficient complementarity to hybridize to the template may be referred to herein as a hybridizing region.

  In other cases, the primers utilized in the methods and compositions described herein may include one or more universal nucleosides. Non-limiting examples of universal nucleosides are 5-nitroindole and inosine described in U.S. Patent Application Publication Nos. 2009/0325169 and 2010/0167353.

  Primers can be designed according to known parameters to avoid secondary structure and self-hybridization. The different primer pairs anneal at about the same temperature, eg, within 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, or 10 ° C of another primer pair. And may be melted. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, 1000, 5000, 10,000 or more primers are used initially. Such primers can hybridize to a target polynucleotide described herein.

Primers can be prepared by a variety of methods, including but not limited to cloning of the appropriate sequence, and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol., 68). Volume: 90 (1979); Brown et al., Methods Enzymol. 68: 109 (1979)). Also, primers can be obtained from commercial sources. Primers may have the same melting temperature. Primers may have non-identical melting temperatures. The length of the primer can be extended or shortened at the 5 ′ or 3 ′ end to produce a primer having a desired melting temperature. One of the primers of the primer pair may be longer than the other primer. Within a primer pair, the 3 'annealing length of the primer may be different. Also, the annealing location of each primer pair can be designed such that the sequence and length of the primer pair provides the desired melting temperature. The formula for determining the melting temperature of primers shorter than 25 base pairs is Wallace's rule (T _M = 2 (A + T) +4 (G + C)). Moreover, you may design a primer using a computer program. The T _M (melting temperature or annealing temperature) of each primer can be calculated using a software program. The annealing temperature of the primer is not limited to these, but is 1, 2, 3, 4, 5th cycle, 6th to 10th cycle, 10th to 15th cycle, 15th to 20th cycle, 20 to 25th cycle, 25 to 25th cycle. It may be recalculated and increased after any amplification cycle, including cycle 30, cycle 30-35, or cycle 35-40. After the first amplification cycle, the 5 'half of the primers may be incorporated into the product from each locus of interest, so the _TM is based on both the 5' and 3 'half sequences of each primer. May be recalculated.

  The primer site includes the region of the template to which the primer hybridizes. In some embodiments, a primer can serve as a starting point for template-directed nucleic acid synthesis. For example, a primer can initiate template-directed nucleic acid synthesis in the presence of four different nucleotides and a polymerizing agent or enzyme such as DNA or RNA polymerase or reverse transcriptase. The primer pair consists of two primers: a first primer having a 5 'upstream region that hybridizes with the 5' end of the template sequence, and a second primer having a 3 'downstream region that hybridizes with the complement of the 3' end of the template sequence. Primers. The primer set comprises two or more primers: a first primer or first plurality of primers having a 5 'upstream region that hybridizes to a 5' end of the template sequence or plurality of template sequences, and a template sequence. Or a second primer or a second plurality of primers having a 3 'downstream region that hybridizes to the complement of the 3' end of the plurality of template sequences. In some embodiments, the primer comprises a target-specific sequence. In some embodiments, the primer comprises a sample barcode sequence. In some embodiments, the primer comprises a universal priming sequence. In some embodiments, the primer comprises a PCR priming sequence. In some embodiments, the primer comprises a PCR priming sequence used to initiate amplification of the polynucleotide. (Dieffenbach, PCR Primer: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, New York, 2003.) The universal primer binding site or sequence allows attachment of the universal primer to polynucleotides and / or amplicons Universal primers are well known in the art and include, but are not limited to, -47F (M13F), alpha MF, AOX3 ', AOX5', BGHr, CMV-30, CMV-50, CVMf, LACrmt, lambda. gt10F, lambda gt10R, lambda gt11F, lambda gt11R, M13 reverse, M13 forward (−20), M13 reverse, mail (male), p10SEQPpQE, pA-120, pet4, pGAP forward, pGLRVpr3, pGLprr2R, pK AC14, pQEFS, pQERS, pucU1, pucU2, reverse A, seqIREStam, seqIRESzpet, seqori, seqPCR, seqpIRES-, seqpIRES +, seqSecTag, seqpSec-Tp, -seqTv, and -seqTv +, seqtromP + As used herein, attachment can refer to covalent and / or non-covalent interactions.The attachment of the universal primer to the universal primer binding site can be accomplished with polynucleotides and polynucleotides. And / or may be used for amplification, detection, and / or sequencing of amplicons. Also about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, It may comprise 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or base pairs.In another example, the universal primer binding site is It includes at least about 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 nucleotides or base pairs. In some embodiments, the universal primer binding site comprises 1-10, 10-20, 10-30, or 10-100 nucleotides or base pairs. In some embodiments, the universal primer binding site is about 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 1-900, 1-800, 1-700, 1 600, 1-500, 1-400, 1-300, 1-200, 1-100, 2-900, 2-800, 2-700, 2-600, 2-500, 2-400, 2-300, 2-200, 2-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-90, 10 80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-10, 5-900, 5- 00, 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 25-900, 25-800, 25-700, 25-600, 25-500, 25-400, 25-300, 25-200, 25- 100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-80 , 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500 -800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900- Contains 1000 nucleotides or base pairs.

A primer may have a length compatible with its use in the synthesis of primer extension products. The primer may be a polynucleotide that is between 8 and 200 nucleotides in length. The length of the primer may depend on the sequence of the template polynucleotide and the template locus. For example, the length and / or melting temperature (T _M ) of a primer or primer set can be optimized. In some cases, the primer has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or More than 60 or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, or less than 60 nucleotides. In some embodiments, the primer is about 8-100 nucleotides in length, for example, 10-75, 15-60, 15-40, 18-30, 20-40, 21-50, 22-45, 25-40, 7-9, 12-15, 15-20, 15-25, 15-30, 15-45, 15-50, 15-55, 15-60, 20-25, 20- 30, 20 to 35, 20 to 45, 20 to 50, 20 to 55, or 20 to 60 nucleotides and any length therebetween. In some embodiments, the primer has a length of at most about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45. , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.

  Generally, one or more pairs of primers can be used in an exponential amplification reaction. One primer of the primer pair may be a forward primer, and one primer of the primer pair may be a reverse primer. In some embodiments, the first pair of primers can be used in an exponential amplification reaction. One primer of the first pair may be a forward primer complementary to the sequence of the first template polynucleotide molecule, and one primer of the first pair may be a first primer of the first template polynucleotide molecule. The second primer may be a reverse primer complementary to the second sequence, and the first template locus may be present between the first sequence and the second sequence. In some embodiments, a second pair of primers can be used in an amplification reaction. One primer of the second pair may be a forward primer complementary to the first sequence of the second target polynucleotide molecule, and one primer of the second pair may be a second target polynucleotide. The second primer may be a reverse primer that is complementary to the second sequence of the molecule, and the second target locus may be between the first and second sequences. In some embodiments, the second target locus comprises a variable light chain antibody sequence. In some embodiments, a third pair of primers can be used in an amplification reaction. One primer of the third pair may be a forward primer complementary to the first sequence of the third template polynucleotide molecule, and one primer of the third pair may be a third template polynucleotide. The third primer may be a reverse primer that is complementary to the second sequence of the molecule, and the third template locus may be located between the first and second sequences.

  One or more primers can anneal to at least a portion of the plurality of template polynucleotides. One or more primers can anneal to the 3 'end and / or 5' end of the plurality of template polynucleotides. One or more primers can anneal to an internal region of the plurality of template polynucleotides. The internal region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 from the 3 'or 5' end of the plurality of template polynucleotides. , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420 , 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 65 , It may be a 700,750,800,850,900 or 1000, nucleotides. The one or more primers may include a panel of primers. One or more primers may include at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more housekeeping gene primers. One or more primers may include a universal primer. The universal primer can anneal to the universal primer binding site. In some embodiments, one or more custom primers anneal to SBCs, target-specific regions, their complements, or any combination thereof. One or more primers may include a universal primer. The one or more primers may be used to amplify primer extension, reverse transcription, linear extension, non-exponential amplification, exponential amplification, PCR, or any other method of amplifying one or more target or template polynucleotides. Or it can be designed to be implemented.

  The target-specific region is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 7 0,800,850,900 or may contain 1000 nucleotides or base pairs. In another example, at least about 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 target-specific regions Of nucleotides or base pairs. In some embodiments, the target-specific region is about 5-10, 10-15, 10-20, 10-30, 15-30, 10-75, 15-60, 15-40, 18-30, 20-40, 21-50, 22-45, 25-40, 7-9, 12-15, 15-20, 15-25, 15-30, 15-45, 15-50, 15-55, 15 60, 20-25, 20-30, 20-35, 20-45, 20-50, 20-55, 20-60, 2-900, 2-800, 2-700, 2-600, 2-500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 25 to 900, 25 to 800, 25 to 700, 25 to 600, 25 to 500, 25 to 400, 25 to 300, 25 to 200, 25 to 25 100, 100-1000, 100-900, 100-800, 00-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200- 400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700- 900, 700-800, 800-1000, 800-90 , Or a 900 to 1000 nucleotides or base pairs.

  Primers can be designed according to known parameters to avoid secondary structure and self-hybridization. In some embodiments, the different primer pairs are at about the same temperature, for example, 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C of another primer pair. Or within 10 ° C. for annealing and melting. In some embodiments, one or more primers of the plurality of primers are at about the same temperature, for example, 1, 2, 3, 4, 5, 6, 7, It may anneal and melt within 8, 9, or 10 ° C. In some embodiments, one or more primers in the plurality may anneal and melt at a different temperature than another primer in the plurality of primers.

  The plurality of primers for one or more steps of the methods described herein can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 , 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18 000, 19,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 50,000,000, 100,000,000 At most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 , 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 , 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 0000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500, 000, 600,000, 700,000, 800,000,000, 900,000,000, 1,000,000, 50,000,000, 100,000,000, or at least about 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 , 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 00, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800, Multiple primers may be included, including 000, 900,000, 1,000,000, 50,000,000, 100,000,000 different primers. For example, each of the plurality of primers may include a different target or template-specific region or sequence.

Reverse transcription In some cases, target polynucleotides are prepared from RNA by reverse transcription. In some cases, a target polynucleotide is prepared from DNA by primer extension, using a polymerase or the like.

  The methods described herein can be used in a coupled reverse transcription-PCR (reverse transcription-PCR). For example, reverse transcription and PCR may be performed in two separate steps. First, a cDNA copy of a sample mRNA may be synthesized using a polynucleotide dT primer, a sequence-specific primer, a universal primer, or any of the primers described herein.

  Reverse transcription and PCR may be performed in a single closed Bessel reaction. For example, three primers may be used, one for reverse transcription and two for PCR. The reverse transcription primer may bind to the mRNA 3 'to the position of the PCR amplicon. Although not required, the reverse transcription primer may include RNA residues or modified analogs such as 2'-O methyl RNA bases that do not form a substrate for RNaseH when hybridized to mRNA.

  The temperature at which the reverse transcription reaction is performed depends on the reverse transcriptase used. In some cases, a thermostable reverse transcriptase is used, and the reverse transcription reaction is carried out at about 37 ° C to about 75 ° C, at about 37 ° C to about 50 ° C, at about 37 ° C to about 55 ° C, at about 37 ° C. C. to about 60.degree. C., about 55.degree. C. to about 75.degree. C., about 55.degree. C. to about 60.degree. C., or about 37.degree. C. to about 60.degree. In some cases, reverse transcriptase is used that transfers three or more non-template terminal nucleotides to the end of the transcript.

  The reverse transcription and PCR reactions described herein can be performed in various formats known in the art, such as in tubes, microtiter plates, microfluidic devices, or preferably in droplets.

  The reverse transcription reaction can be performed in a volume ranging from 5 μL to 100 μL, or in a reaction volume of 10 μL to 20 μL. For droplets, the reaction volume may range from 1 pL to 100 nL, or from 10 pL to 1 nL. In some cases, the reverse transcription reaction is performed in droplets having a volume of about 1 nL or less. In some cases, the PCR reaction is in a droplet having a reaction volume range of 1 pL to 100 nL, preferably 10 pL to 1 nL. In some cases, the PCR reaction is performed in droplets having a volume of about 1 nL or less. In some cases, the reverse transcription and PCR reactions are performed in the same droplet with a reaction volume range of 1 pL to 100 nL, or 10 pL to 1 nL. In some cases, the reverse transcription and PCR reactions are performed in droplets having a volume of about 1 nL or less, or about 1 pL or less. In some cases, the reverse transcription reaction and the PCR reaction are performed in different droplets. In some cases, the reverse transcription reaction and the PCR reaction are performed in multiple droplets, each having a reaction volume range of 1 pL to 100 nL, or 10 pL to 1 nL. In some cases, the reverse transcription reaction and the PCR reaction are performed in multiple droplets each having a volume of about 1 nL or less.

  In some cases, the first PCR reaction is in a first drop having a reaction volume of 1 pL to 100 nL, preferably in the range of 10 pL to 1 nL, and the second PCR reaction is 1 pL to 100 nL. , Preferably in a second droplet having a reaction volume in the range of 10 pL to 1 nL. In some cases, the first PCR reaction is in a first droplet having a volume of about 1 nL or less, and the second PCR reaction is in a second droplet having a volume of about 1 nL or less. Within the droplet.

  In some cases, the first PCR reaction and the second PCR reaction are performed in a plurality of droplets each having a reaction volume range of 1 pL to 100 nL, or 10 pL to 1 nL. In some cases, the first and second PCR reactions are performed in a plurality of droplets each having a volume of about 1 nL or less.

  A target polynucleotide, such as RNA, may be reverse transcribed into cDNA using one or more reverse transcription primers. One or more reverse transcription primers may include a region that is complementary to a region of the RNA, such as a constant region (eg, a heavy or light chain constant region of an mRNA or a poly-A tail). In some embodiments, the reverse transcription primer has a first reverse transcription primer having a region complementary to the constant region of the first RNA, and a second reverse transcription primer having a region complementary to the constant region of the second RNA. May be included. In some embodiments, the reverse transcription primer is a first reverse transcription primer having a region complementary to the constant region of the first RNA, and a region complementary to the constant region of one or more RNAs, respectively. May comprise one or more reverse transcription primers having

  In some embodiments, the reverse transcription primer does not include a barcode.

  The reverse transcription primer may further include a region that is not complementary to a region of the RNA. In some embodiments, the region that is not complementary to a region of the RNA is 5 'to a region of the primer that is complementary to the RNA. In some embodiments, the region that is not complementary to a region of the RNA is 3 'to a region of the primer that is complementary to the RNA. In some embodiments, the region that is not complementary to a region of the RNA is a 5 'overhang region. In some embodiments, regions that are not complementary to regions of the RNA include priming sites for amplification and / or sequencing reactions. Using one or more primers described herein, the RNA molecule is reverse transcribed using suitable reagents known in the art.

  After performing a reverse transcription reaction of the RNA molecule, the resulting cDNA molecule is barcoded with a molecular barcode and a Bessel barcode, and one or more PCR reactions, such as a first and / or a second PCR reaction. For amplification. The first and / or second PCR reactions may utilize a single pair of primers or multiple primer pairs. The first and / or second PCR reactions may utilize multiple forward / reverse and reverse primers. The first and / or second PCR reactions may utilize multiple forward / reverse primers and forward primers. The first and / or second primer of the plurality of forward / reverse primers may be a forward / reverse primer containing a region complementary to a cDNA molecule or a barcoded cDNA molecule. The first and / or second primer of the plurality of forward / reverse primers may be a forward / reverse primer containing a region complementary to a barcoded cDNA molecule.

  In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, each of the forward / reverse primers of the plurality of forward / reverse primers comprising a cDNA or V-coded barcoded cDNA. Includes a region that is complementary to one or more upstream or downstream regions of the segment. For example, the plurality of forward / reverse primers may include a forward / reverse primer that includes a region complementary to the upstream or downstream region of the V segment of the cDNA or barcoded cDNA, and one or more of the V segments of the cDNA or barcoded cDNA. Includes one or more other forward / reverse primers that include regions complementary to multiple other upstream or downstream regions. For example, the plurality of forward / reverse primers may include a first and / or second forward / reverse primer that includes a region complementary to a first and / or second upstream or downstream region of a V segment of a cDNA or barcoded cDNA. Primers, and a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the V segment of the cDNA or barcoded cDNA. For example, the plurality of forward / reverse primers may include a first and / or second forward / reverse primer that includes a region complementary to a first and / or second upstream or downstream region of a V segment of a cDNA or barcoded cDNA. A primer, a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the V segment of the cDNA or barcoded cDNA, and a third upstream or reverse of the V segment of the cDNA or barcoded cDNA. And a third forward / reverse primer containing a region complementary to the downstream region. Primers in multiple forward / reverse primers can be used to anneal to any possible upstream or downstream region of any V segment expressed by cells such as immune B cells or T cells in a sample.

  In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers comprises a cDNA or a barcoded cDNA. It contains regions complementary to one or more upstream or downstream regions of the C segment. For example, the plurality of forward / reverse primers include a forward / reverse primer that includes a region complementary to the upstream or downstream region of the C segment of the cDNA or barcoded cDNA, and one or more of the C segment of the cDNA or barcoded cDNA. Includes one or more other forward / reverse primers that include regions complementary to multiple other upstream or downstream regions. For example, the plurality of forward / reverse primers comprises a first and / or second forward / reverse primer comprising a region complementary to a first and / or second upstream or downstream region of the C segment of the cDNA or barcoded cDNA. Primers and a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the C segment of the cDNA or barcoded cDNA. For example, the plurality of forward / reverse primers comprises a first and / or second forward / reverse primer comprising a region complementary to a first and / or second upstream or downstream region of the C segment of the cDNA or barcoded cDNA. A primer, a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the C segment of the cDNA or barcoded cDNA, and a third upstream or reverse of the C segment of the cDNA or barcoded cDNA. And a third forward / reverse primer containing a region complementary to the downstream region. Primers in multiple forward / reverse primers can be used to anneal to any possible upstream or downstream region of any C segment expressed by cells such as immune B cells or T cells in a sample.

  In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers comprises a molecular bar of the barcoded cDNA. It contains regions complementary to one or more upstream or downstream regions of the code. For example, the plurality of forward / reverse primers may include a forward / reverse primer that includes a region complementary to an upstream or downstream region of the barcoded cDNA molecular barcode, and one or more of the barcoded cDNA molecular barcodes. Includes one or more other forward / reverse primers that include regions complementary to other upstream or downstream regions. For example, the plurality of forward / reverse primers may be first and / or second forward / reverse primers comprising regions complementary to first and / or second upstream or downstream regions of the molecular barcode of the barcoded cDNA. And a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the molecular barcode of the barcoded cDNA. For example, the plurality of forward / reverse primers may be first and / or second forward / reverse primers comprising regions complementary to first and / or second upstream or downstream regions of the molecular barcode of the barcoded cDNA. A second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the molecular barcode of the barcoded cDNA, and a third upstream or downstream region of the molecular barcode of the barcoded cDNA. And a third forward / reverse primer containing a complementary region. Multiple forward / reverse primers can be used to anneal to any possible upstream or downstream region of any molecular barcode expressed by cells such as immune B cells or T cells in a sample.

  In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers is a vessel bar of the barcoded cDNA. It includes regions complementary to one or more upstream or downstream regions of the code. For example, the plurality of forward / reverse primers may include a forward / reverse primer that includes a region complementary to a region upstream or downstream of the Bessel barcode of the barcoded cDNA, and one or more of the Bessel barcode of the barcoded cDNA. Includes one or more other forward / reverse primers that include regions complementary to other upstream or downstream regions. For example, the plurality of forward / reverse primers comprises a first and / or second forward / reverse primer comprising a region complementary to the first and / or second upstream or downstream region of the Bessel barcode of the barcoded cDNA. And a second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the Bessel barcode of the barcoded cDNA. For example, the plurality of forward / reverse primers comprises a first and / or second forward / reverse primer comprising a region complementary to the first and / or second upstream or downstream region of the Bessel barcode of the barcoded cDNA. A second forward / reverse primer comprising a region complementary to a second upstream or downstream region of the Bessel barcode of the barcoded cDNA, and a third upstream or downstream region of the Bessel barcode of the barcoded cDNA. And a third forward / reverse primer containing a complementary region. Primers in multiple forward / reverse primers can be used to anneal to any possible upstream or downstream region of any vessel barcode expressed by cells such as immune B cells or T cells in a sample.

  The forward / reverse primer in the plurality of forward / reverse primers further comprises a region that is not complementary to a region of the RNA. In some embodiments, the region that is not complementary to a region of the RNA is 5 'to the region of the forward / reverse primer that is complementary to the RNA (i.e., the region upstream or downstream of the V segment). In some embodiments, the region that is not complementary to a region of the RNA is 3 'to a region of the forward / reverse primer that is complementary to the RNA. In some embodiments, the region that is not complementary to a region of the RNA is a 5 'overhang region. In some embodiments, the region that is not complementary to a region of the RNA comprises a priming site for an amplification and / or a second sequencing reaction. In some embodiments, the region that is not complementary to a region of the RNA comprises a priming site for an amplification and / or a third sequencing reaction. In some embodiments, the regions that are not complementary to regions of the RNA include priming sites for the second and third sequencing reactions. In some embodiments, the sequences of the priming sites for the second and third sequencing reactions are the same. One or more forward / reverse and reverse primers described herein are used to amplify the cDNA molecule using suitable reagents known in the art. In some embodiments, the region is complementary to a region of the RNA, such as a constant region of the mRNA or a poly-A tail.

In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers has an affinity-oligonucleotide conjugate Alternatively, it comprises a region complementary to one or more upstream or downstream regions of the vessel barcode of the barcode oligonucleotide of the tetramer-oligonucleotide conjugate. In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers has an affinity-oligonucleotide conjugate Alternatively, it comprises a region complementary to one or more upstream or downstream regions of the AID of the barcode oligonucleotide of the tetramer-oligonucleotide conjugate. In some embodiments, the plurality of forward / reverse primers comprises one or more forward / reverse primers, wherein each of the forward / reverse primers in the plurality of forward / reverse primers has an affinity-oligonucleotide conjugate Alternatively, the barcoding oligonucleotide of the tetramer-oligonucleotide conjugate comprises a region complementary to one or more upstream or downstream regions of the AMB. For example, the multiple forward / reverse primers may be complementary to the upstream or downstream region of the vessel-barcode, AID, and / or AMB of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate barcoding oligonucleotide. Forward / reverse primer comprising a specific region and one or more of the vessel-barcode, AID and / or AMB of the affinity-oligonucleotide or tetramer-oligonucleotide conjugate barcoding oligonucleotide And one or more other forward / reverse primers that include regions complementary to other upstream or downstream regions. For example, the plurality of forward / reverse primers may be the first and / or the first and / or the second of a vessel barcode, AID, and / or AMB of an affinity-oligonucleotide or tetramer-oligonucleotide conjugate barcoding oligonucleotide. First and / or second forward / reverse primers comprising a region complementary to the two upstream or downstream regions, and a bar-coded oligo of an affinity-oligonucleotide or tetramer-oligonucleotide conjugate A second forward / reverse primer comprising a region complementary to the second upstream or downstream region of the vessel barcode, AID, and / or AMB of nucleotides. For example, the plurality of forward / reverse primers may be the first and / or the first and / or the second of a vessel barcode, AID, and / or AMB of an affinity-oligonucleotide or tetramer-oligonucleotide conjugate barcoding oligonucleotide. A first and / or a second forward / reverse primer, an affinity-oligonucleotide conjugate or a tetramer-oligonucleotide conjugate, comprising a region complementary to the two upstream or downstream regions, A second forward / reverse primer comprising a region complementary to the second upstream or downstream region of the vessel barcode, AID and / or AMB, and an affinity-oligonucleotide conjugate or tetramer-o Including Gore nucleotides conjugated barcoding vessel barcode oligonucleotides, AID, and / or the like third forward / reverse primer comprising a third region complementary to the upstream or downstream region of the AMB.
amplification

  After adding the vessel barcode to the target polynucleotide, the target polynucleotide can be amplified. For example, the oligonucleotide can be amplified after the Bessel barcode is added to the affinity-oligonucleotide or tetramer-oligonucleotide conjugate oligonucleotide. For example, a Bessel barcoded cell polynucleotide can be amplified after adding a Bessel barcode to the cell polynucleotide.

  The amplification reaction may include one or more additives. In some cases, the one or more additives are dimethylsulfoxide (DMSO), glycerol, betaine (mono) hydrate (N, N, N-trimethylglycine = carboxy-methyl] trimethylammonium, trehalose, 7 -Deaza-2'-deoxyguanosine triphosphate (dC7GTP or 7-deaza-2'-dGTP), BSA (bovine serum albumin), formamide (methaneamide), tetramethylammonium chloride (TMAC), other tetraalkylammonium derivatives (Eg, tetraethylammonium chloride (TEA-Cl) and tetrapropylammonium chloride (TPrA-Cl)), nonionic detergents (eg, Triton X-100, Tween 20, Nonidet P-40 (NP-40)), Or PREXCEL-Q. In some cases, the amplification reaction includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different additives. In some cases, the amplification reaction includes at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different additives.

  A thermocycling reaction can be performed on a sample contained in a reaction volume (eg, droplets). The droplets may be polydispersed, or preferably monodispersed, and those skilled in the art will be able to stir, sonicate, or microfluidically pass through the T-channel junction; Or it can be generated by other means. The density may be greater than 20,000 drops / 40 ul (1 nL drop), 200,000 drops / 40 ul (100 pL drop). Droplets can maintain integrity during thermal cycling. Droplets are about 10,000 drops / μL, 100,000 drops / μL, 200,000 drops / μL, 300,000 drops / μL, 400,000 drops / μL, 500,000 drops / ΜL, 600,000 droplets / μL, 700,000 droplets / μL, 800,000 droplets / μL, 900,000 droplets / μL, or at a density greater than 1,000,000 droplets / μL; Integrity can be maintained during thermal cycling. In other cases, two or more droplets do not aggregate during thermal cycling. In other cases, more than 100 or more than 1,000 droplets do not aggregate during thermal cycling.

  Although not limited to these, coli DNA polymerase, E. coli. catalyze primer extension, including the Klenow fragment of E. coli DNA polymerase 1, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase, Pfu DNA polymerase, Vent DNA polymerase, bacteriophage 29, REDTaq ™, genomic DNA polymerase, or sequencenase. Any DNA polymerase can be used. In some cases, a thermostable DNA polymerase is used. Alternatively, the reaction may be heated to 95 ° C. for 2 minutes before adding the polymerase, or a hot start PCR may be performed which allows the polymerase to be inactive until the first heating step of the first cycle. The use of hot start PCR can minimize non-specific amplification. Any number of PCR cycles, eg, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 Cycles, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or more than 45 times Cycle or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, DNA can be amplified using less than 40, 41, 42, 43, 44, or 45 cycles. The number of amplification cycles is about 1-45, 10-45, 20-45, 30-45, 35-45, 10-40, 10-30, 10-25, 10-20, 10-15, 20-35. , 25-35, 30-35, or 35-40.

  Amplification of the target nucleic acid can be performed by any means known in the art. The target nucleic acid may be amplified by polymerase chain reaction (PCR) or isothermal DNA amplification. Examples of PCR techniques that can be used include, but are not limited to, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real-time PCR (reverse transcription-PCR), simple PCR. One-cell PCR, restriction fragment length polymorphism PCR (PCR-RFLP), PCR-RFLP / reverse transcription-PCR-RFLP, hot start PCR, nested PCR, in situ polony PCR, in situ rolling circle amplification (RCA), digital PCR (DPCR), droplet digital PCR (ddPCR), bridge PCR, picoliter PCR, and emulsion PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcription amplification, molecular inversion probe (MIP) PCR, self-sustaining sequence replication, selective amplification of target polynucleotide sequences, consensus sequence primed Polymerase chain reaction (CP-PCR; consensus sequence primed polymerase chain reaction), arbitrary primed polymerase chain reaction (AP-PCR), degenerate polynucleotide primed PCR (DOP-PCR; degenerate polynucleotide-primed PCR), and a nucleic acid-based sequence amplification method (NABSA; nucleic acid based sequence amplification). Other amplification methods that can be used herein include U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and 6,582,938. And Qbeta replicase-mediated RNA amplification methods. The amplification may be an isothermal amplification, for example, an isothermal linear amplification.

  In some embodiments, amplification does not occur on a solid support. In some embodiments, amplification does not occur on the solid support within the droplet. In some embodiments, if the amplification is not in droplets, the amplification occurs on a solid support.

Sequencing After performing one or more of the methods or method steps described herein, the resulting library of polynucleotides may be sequenced.

  Sequencing can be performed by any sequencing method known in the art. In some embodiments, sequencing can be performed at high throughput. Suitable next-generation sequencing technologies include 454 Life Science Platform (Roche, Branford, Conn.) (Margulies et al., Nature, 437, 376-380 (2005)); Illumina Genome Analyzer, GoldenGate Methylation. Assays, or Infinium methylation assays, i.e., Infinium HumanMethylation 27K BeadArray, or VeraCode GoldenGate Methylation Array (Illumina, San Diego, Calif., Bibkova et al., Bibkova et al., Genome Res. 16, 383-393 (2006) and US Patent 2006). 6,306,597, 7,598,035, 7,232,656) or ligation DNA sequencing by the SOLiD system (Applied Biosystems / Life Technologies, U.S. Patent Nos. 6,797,470, 7,083,917, 7,166,434, 7,320,865, 7). No., 332,285, 7,364,858, and 7,429,453); or Helicos True Single Molecular DNA sequencing technology (Harris et al., Science, 320, 106-109 (2008)). And US Patent Nos. 7,037,687, 7,645,596, 7,169,560, and 7,769,400), Pacific Biosciences Single-Molecule Real-Time (SMRTTTm) technology and sequence. Determination method (Soni et al., Clin. Chem., 3, pp. 1996 to 2001 (2007)), and the like. Such systems allow multiplex parallel sequencing of large numbers of polynucleotides isolated from a sample (Dear, Brief Funct. Genomic Proteomic, 1 (4), 397-416 (2003), and McCaughan et al., J. Pathol., 220, 297-306 (2010)). In some embodiments, polynucleotides are sequenced by ligation of dye-modified probes, pyrosequencing, or single molecule sequencing. Polynucleotide sequence determinations include Helioscope ™ single molecule sequencing, nanopore DNA sequencing, Lynx Therapeutics massively parallel signature sequencing (MPSS), 454 pyrosequencing, single molecule real-time (RNAP) sequencing Sequencing, Illumina (Solexa) Sequencing, SOLiD Sequencing, Ion Torrent ™, Ion Semiconductor Sequencing, Single Molecule SMRT ™ Sequencing, Polony Sequencing, DNA Nanoball Sequencing, and It can be carried out by a sequencing method such as the VisiGen Biotechnology technique. Alternatively, for determining the sequence of a polynucleotide, one molecule real-time (such as, but not limited to, the genome analyzers IIx, HiSeq, and MiSeq provided by Illumina; the PacBio RS system and the Solexa sequencer provided by Pacific Biosciences, California) SMRT ™ technology; Helicos Inc. A sequencing platform may be used, including True Single Molecular Sequencing (tSMS ™) technology, such as HeliScope ™ provided by (Cambridge, Mass.). Sequencing may include MiSeq sequencing. Sequencing may include HiSeq sequencing. In some embodiments, determining the sequence of the polynucleotide comprises paired-end sequencing, nanopore sequencing, high-throughput sequencing, shotgun sequencing, dye terminator sequencing, multiple primer DNA sequencing, Includes primer walking, Sanger dideoxy sequencing, Maxim-Gilbert sequencing, pyrosequencing, true single molecular sequencing, or any combination thereof. Alternatively, the sequence of the polynucleotide may be determined by electron microscopy or a chemosensitive field effect transistor (chemFET) array.

  The method may further include sequencing one or more polynucleotides in the library. The method may further include aligning one or more polynucleotide sequences, sequence reads, amplicon sequences, or amplicon set sequences in the library with each other.

  Alignment may include comparing a test sequence, such as a sequence read, with one or more other test sequences, reference sequences, or combinations thereof. In some embodiments, alignments may be used to determine a consensus sequence from multiple or aligned sequences. In some embodiments, the alignment comprises determining a consensus sequence from multiple sequences each having the same molecular barcode or Bessel barcode. In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, of the length of the reference sequence. 90%, or at least 95%. The actual comparison of two or more sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). . Such algorithms are incorporated in the NBLAST and XBLAST programs (version 2.0) described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (eg, NBLAST) may be used. For example, the parameters for sequence comparison may be set to score = 100, wordlength = 12, or may vary (eg, W = 5 or W = 20). Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In some embodiments, percent identity between two amino acid sequences can be achieved, for example, using the GAP program of the GCG software package (Accelrys, Cambridge, UK).

  Sequencing may include sequencing at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides or base pairs of the polynucleotide. Good. In some embodiments, the sequencing comprises sequencing at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or base pairs of the polynucleotide. including. In other cases, sequencing comprises sequencing at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more nucleotides or base pairs of the polynucleotide. Including doing.

  Sequencing may include at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more sequencing reads per run. As used herein, a sequence read includes a sequence of nucleotides determined from a sequence or stream of data generated by a sequencing technique. In some embodiments, sequencing comprises at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more sequencing reads per run. Including. Sequencing can result in more than about 1,000,000, less than about 1,000,000, or equal to about 1,000,000 sequencing reads per run. May be included. Sequencing may include more than about 200,000,000, less than about 200,000,000, or equal to about 200,000,000 sequencing reads per run.

Enzymes The methods and kits disclosed herein may include one or more enzymes. Examples of enzymes include, but are not limited to, ligases, reverse transcriptases, polymerases, and restriction nucleases.

  In some embodiments, attaching the adapter to the polynucleotide comprises using one or more ligases. Examples of ligases include, but are not limited to, DNA ligases such as DNA ligase I, DNA ligase III, DNA ligase IV, and T4 DNA ligase; and RNA ligases such as T4 RNA ligase I and T4 RNA ligase II. .

  The methods and kits disclosed herein may further include using one or more reverse transcriptases. In some embodiments, the reverse transcriptase is HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, and telomerase reverse transcriptase. In some embodiments, the reverse transcriptase is M-MLV reverse transcriptase.

  In some embodiments, the methods and kits disclosed herein involve using one or more proteases.

  In some embodiments, the methods and kits disclosed herein involve using one or more polymerases. Examples of polymerases include, but are not limited to, DNA polymerase and RNA polymerase. In some embodiments, the DNA polymerase is DNA polymerase I, DNA polymerase II, DNA polymerase III holoenzyme, and DNA polymerase IV. Commercially available DNA polymerases include, but are not limited to, Bst2.0 DNA polymerase, Bst2.0 WarmStart ™ DNA polymerase, Bst DNA polymerase, Sulfolobus DNA polymerase IV, Taq DNA polymerase, 9 ° N ™ ) MDNA polymerase, Deep VentR ™ (exo) DNA polymerase, Deep VentR ™ DNA polymerase, Hemo KlenTaq ™, LongAmp ® Taq DNA polymerase, OneTaq ® DNA polymerase, Phusion ® ) DNA polymerase, Q5 ™ high fidelity DNA polymerase, Therminator ™ γ DNA polymerase Therminator ™ DNA polymerase, Therminator ™ II DNA polymerase, Therminator ™ III DNA polymerase, VentR ™ DNA polymerase, VentR ™ (exo) DNA polymerase, Bsu DNA polymerase, phi29 DNA polymerase , T4 DNA polymerase, T7 DNA polymerase, terminal transferase, Titanium® Taq polymerase, KAPA Taq DNA polymerase, and KAPA Taq hot start DNA polymerase.

  In some embodiments, the polymerase is RNA polymerase I, RNA polymerase II, RNA polymerase III, E. coli. RNA polymerases such as E. coli poly (A) polymerase, phi6 RNA polymerase (RdRP), poly (U) polymerase, SP6 RNA polymerase, and T7 RNA polymerase.

Additional Reagents The methods and kits disclosed herein may include using one or more reagents. Examples of reagents include, but are not limited to, PCR reagents, ligation reagents, reverse transcription reagents, enzyme reagents, hybridization reagents, sample preparation reagents, affinity capture reagents, solid supports such as beads, and nucleic acid purification and / or Or a reagent for isolation.

  In other embodiments, the methods, kits, and compositions disclosed herein may include a support. In some embodiments, the methods, kits, and compositions disclosed herein do not include a support. Typically, a solid support includes one or more materials that include one or more rigid or semi-rigid surfaces. In some embodiments, the support is a non-solid support. The support or substrate may include a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. In some embodiments, one or more surfaces of the support are substantially flat, but in some embodiments, for example, have wells, raised areas, pins, or etched grooves, etc. It may be desirable for the synthetic regions for the various compounds to be physically separated. In some embodiments, the solid support comprises beads, resins, gels, microspheres, or other geometries. Alternatively, solid supports may include silica chips, microparticles, nanoparticles, plates, and arrays. Solid supports may involve the use of self-assembling beads in microwells. For example, solid supports include Illumina's BeadArray technology. Alternatively, the solid support includes Abbott Molecular's Bead Array technology and Applied Microarray's FlexiPlex ™ system. In another example, the solid support is a plate. Examples of plates include, but are not limited to, MSD multi-array plate, MSD Multi-Spot® plate, microplate, ProteOn microplate, AlphaPlate, DELFIA plate, IsoPlate, and LumaPlate. In some embodiments, the support may include a plurality of beads. In some embodiments, the support may include an array. In some embodiments, the support may include a glass slide. Methods, substrates, and techniques applicable to polymers (US Pat. Nos. 5,744,305, 5,143,854, 5,242,974, 5,252,743, 5,324). No. 5,633, No. 5,384,261, No. 5,405,783, No. 5,424,186, No. 5,451,683, No. 5,482,867, No. 5,491,074. No. 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, Nos. 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, and 5th No. 5,858,659, No. 5,936,324, No. 5,968,7. No. 5, No. 5,974,164, No. 5,981,185, No. 5,981,956, No. 6,025,601, No. 6,033,860, No. 6,040,193 6,090,555, 6,136,269, 6,269,846, and 6,428,752; U.S. Patent Application Publication Nos. 20090149340, 20080038559, 20050074787; PCT Publication Nos. WO00 / 58516, WO99 / 36760, and WO01 / 58593). Attachment of the polynucleotide to the support may include amine-thiol bridges, maleimide bridges, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide, Zenon, or SiteClick. Attachment of the labeled nucleic acid to the support may include attaching biotin to the plurality of polynucleotides, and coating one or more beads with streptavidin. In some embodiments, the support is a bead. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugate beads (eg, anti-immunoglobulin microbeads), Protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, polynucleotide dT conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads , And BcMag ™ carboxy-terminated magnetic beads. The diameter of the beads may be about 5 μm, 10 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm. The solid support may be an array or a microarray. The solid support may include discrete regions. The solid support may be an array, for example an addressable array.

  The solid support can include virtually any insoluble or solid material, and a solid support composition that is insoluble in water is often selected. For example, solid supports include silica gel, glass (eg, controlled pore glass (CPG)), nylon, Sephadex®, Sepharose®, cellulose, metal surfaces (eg, steel, gold, silver, aluminum). , Silicon, and copper), magnetic materials, plastic materials (eg, polyethylene, polypropylene, polyamide, polyester, and polyvinylidene difluoride (PVDF)), and the like, or may consist essentially of these. Good. Examples of beads used according to this embodiment include affinity moieties that allow interaction between the beads and the nucleic acid molecule. The solid phase (eg, beads) may include a member of a binding pair (eg, avidin, streptavidin, or a derivative thereof). For example, the beads may be streptavidin-coated beads, and the nucleic acid molecules immobilized on the beads may include a biotin moiety. In some cases, each polynucleotide molecule may include two affinity moieties, such as biotin, to further stabilize the polynucleotide. The beads may contain additional features that can be used for immobilizing nucleic acids or used in downstream screening or selection processes. For example, the beads may include an affinity moiety, a fluorescent label, or a fluorescent quencher. In some cases, the beads may be magnetic. In some cases, the support is a bead. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugate beads (eg, anti-immunoglobulin microbeads), Protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, polynucleotide dT conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads , And BcMag ™ carboxy-terminated magnetic beads. The beads or particles may be swellable (eg, polymeric beads such as Wang resin) or non-swellable (eg, CPG). In some embodiments, the solid phase is substantially hydrophilic. In some embodiments, the solid phase (eg, beads) is substantially hydrophobic. In some embodiments, the solid phase comprises a binding pair member (eg, avidin, streptavidin, or a derivative thereof) and is substantially hydrophobic or substantially hydrophilic. In some embodiments, the solid phase comprises a member of a binding pair (eg, avidin, streptavidin, or a derivative thereof) and has more than about 1350 picomoles of free capture agent (eg, free biotin) per mg of solid support. Also have high binding capacity. In some embodiments, the binding capacity of the solid phase comprising the members of the binding pair is 800, 900, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600 per mg of solid support. Higher than 1800, 2000 picomoles of free scavenger. Other examples of beads suitable for the present invention are colloidal gold or beads such as polystyrene beads or silica beads. Virtually any bead radius can be used. Examples of beads include beads having a radius ranging from 150 nanometers to 10 microns. Other sizes can be used.

  The methods and kits disclosed herein may include using one or more buffers. Examples of buffers include, but are not limited to, wash buffers, ligation buffers, hybridization buffers, amplification buffers, and reverse transcription buffers. In some embodiments, the hybridization buffer is a commercially available buffer such as a TMAC Hyb solution, an SSPE hybridization solution, and an ECONO ™ hybridization buffer. The buffers disclosed herein may include one or more detergents.

  The methods and kits disclosed herein may include using one or more carriers. The carrier can enhance or enhance the efficiency of one or more of the reactions disclosed herein (eg, ligation reaction, reverse transcription, amplification, hybridization). The carrier can reduce or prevent loss of non-specificity of the molecules or any of their products (eg, polynucleotides and / or amplicons). For example, the carrier can reduce loss of non-specificity of the polynucleotide due to absorption on the surface. The carrier can reduce the affinity of the polynucleotide for the surface or substrate (eg, container, Eppendorf tube, pipette tip). Alternatively, the carrier can increase the affinity of the polynucleotide for a surface or substrate (eg, beads, arrays, glasses, slides, chips). The carrier can prevent degradation of the polynucleotide. For example, a carrier can protect an RNA molecule from ribonuclease. Alternatively, the carrier can protect the DNA molecule from DNase. Examples of carriers include, but are not limited to, polynucleotides such as DNA and / or RNA, or polypeptides. Examples of DNA carriers include plasmids, vectors, polyadenylated DNA, and DNA polynucleotides. Examples of RNA carriers include polyadenylated RNA, phage RNA, phage MS2 RNA, E. coli. coli RNA, yeast RNA, yeast tRNA, mammalian RNA, mammalian tRNA, short polyadenylated synthetic ribonucleotides, and RNA polynucleotides. The RNA carrier may be a polyadenylated RNA. Alternatively, the RNA carrier may be a non-polyadenylated RNA. In some embodiments, the carrier is from a bacterium, yeast, or virus. For example, the carrier may be a polynucleotide or polypeptide derived from a bacterium, yeast, or virus. For example, the carrier is a protein derived from Bacillus subtilis. In another example, the carrier is a polynucleotide from Escherichia coli. Alternatively, the carrier is a polynucleotide or peptide from a mammal (eg, human, mouse, goat, rat, cow, sheep, pig, dog, or rabbit), bird, amphibian, or reptile.

  The methods and kits disclosed herein may include using one or more control substances. Control substances can include control polynucleotides, inactive enzymes, non-specific competitors. Alternatively, control substances include bright hybridization, bright probe controls, nucleic acid templates, spike-in controls, PCR amplification controls. The PCR amplification control may be a positive control. In another example, the PCR amplification control is a negative control. The nucleic acid template control may be at a known concentration. The control substance may include one or more labels.

  The spike-in control may be a template added to the reaction or sample. For example, a spike-in template may be added to the amplification reaction. The spike-in template may be added to the amplification reaction at any time after the first amplification cycle. In some embodiments, the spike-in template has a cycle number of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35. , 40, 45, or 50 times before being added to the amplification reaction. The spike-in template may be added to the amplification reaction at any time before the last amplification cycle. A spike-in template may include one or more nucleotides or nucleobase pairs. The spike-in template may include DNA, RNA, or any combination thereof. The spike-in template may include one or more labels.

  What is disclosed herein can be used with the methods and compositions disclosed herein, and can be used with the methods and compositions to prepare the methods and compositions. Branches, materials, compositions and components or products of methods and compositions that can be used. Where combinations, subsets, interactions, groups, etc. of these substances are disclosed, where specific references to various individual and collective combinations and permutations of these molecules and compounds cannot be explicitly disclosed. Nevertheless, it is understood that each is specifically contemplated and described herein. For example, if a nucleotide or nucleic acid is disclosed and discussed, and some modification that can be made to some molecule containing the nucleotide or nucleic acid is discussed, the nucleotide or nucleic acid unless otherwise indicated. Any and all combinations and permutations and possible modifications are specifically contemplated. This concept applies to all aspects of the present application, including but not limited to methods for making and using the methods and compositions of the present disclosure. Thus, if there are various additional steps that can be performed, each such additional step can be performed in any particular embodiment or combination of embodiments of the disclosed method, and It is understood that such combinations are to be specifically contemplated and considered to be disclosed.

  Although some embodiments described herein have been shown and described herein, such embodiments are provided by way of example only. Now, those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the disclosure provided herein. It should be understood that various alternatives to the embodiments described herein may be used in performing the methods described herein.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references include embodiments of the methods and compositions that can be used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition, published from Merck Research Laboratories, 2006 (ISBN 0-9119102). Published by Benjamin Lewin, Genes IX, Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); published by Kendrew et al. (Eds.), The Encyclopedia of Mol. Biology, Blackwell Science Ltd., 1994 (ISBN 0-632). -02182-9); and Robert A. Meyers (eds.), Mol. Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

  Standard procedures of the present disclosure are described below: for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1982); Sambrook et al., Molecular Cloning. A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986). Or Methods in Enzymology: Guide to Molecular Cloning Techniques, Volume 152, SL Berger and AR Kimmerl (eds.), Academic Press Inc., San Diego, USA (1987). Current Protocols in Molecular Biology (CPMB) (edited by Fred M. Ausubel et al., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (edited by John E. Coligan et al., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI) (edited by John E. Coligan et al., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (edited by Juan S. Bonifacino et al., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique, by R. Ian Freshney, publisher: Wiley-Liss; 5th edition (2005), and Animal Cell Culture Methods (Methods in Cell Biology, vol. 57, Jennie P. Mather and David Barnes, Ed., Academic Press, First Edition, 1998).

(Example 1)
Immunotyping of single T lymphocytes in T cell populations The immunophenotyping method described herein was validated by analyzing both CD4 and CD8 mRNA and surface protein expression in human T lymphocytes. (FIG. 1). Typically, mature T cells are expected to be of either the CD4 or CD8 subtype. The CD4 subtype should express both CD4 mRNA and protein, but should express either CD8 mRNA or protein. The CD8 subtype should express both CD8 mRNA and protein, but should express either CD4 mRNA or protein.

  30,000 T cells were incubated with both a CD4 specific antibody-oligonucleotide containing the CD4 antigen ID sequence and a CD8 specific antibody-oligonucleotide containing the CD8 antigen ID sequence. T cell CD4, CD8, and TCR mRNA content were also analyzed in single cell emulsion experiments. T cell receptor alpha, T cell receptor beta, CD4 and CD8 mRNAs are reverse transcribed into cDNA and droplet specific barcode sequences fused to both cDNA and antibody conjugated oligonucleotides by polymerase chain reaction did. This DNA was extracted from the emulsion and analyzed by the next generation sequencing method.

  Almost all droplet barcodes (associated with a single cell) were associated with either a CD4-specific or a CD8-specific antigen ID. Substantial agreement was found between mRNA and assignment based on surface proteins (FIG. 3).

(Example 2)
Sequencing of the immune repertoire has broad applications in basic immunology, autoimmunity, infectious diseases, and oncology. Many studies have investigated the circulating BCR and TCR diversity, but there has been increasing interest in TIL immunoreceptors. Its function is highly associated with cancer growth or regression, but to varying degrees and often uncharacterized. Important for a better understanding of TIL is the recovery and functional characterization of TIL BCRs and TCRs, as they may allow the identification of new tumor-associated antigens. Tumor antigens are critically needed to develop cancer vaccines, understand the role of checkpoint inhibitors, and advance chimeric antigen receptor T cell (CAR-T) therapy for solid tumors It has been. However, despite decades of technical advances in immunosequencing, full-length native paired BCRs (heavy and light chains) and TCRs (alpha and beta chains) limit the observed repertoire. No studies have been recovered from heterogeneous samples, such as tumors, without performing in vitro culture or cell sorting, and biasing. Primary uncultured immune cells are very technically difficult because they can contain less than one-hundredth of the receptor RNA of in vitro stimulated cells. Microfluidic emulsion-based method for parallel isolation and DNA barcoding of large numbers of single cells to enable comprehensive analysis of native paired BCRs and TCRs from complex heterogeneous samples Was developed. Up to one million cells / hour are isolated in individual approximately 65 picoliter emulsion droplets. The cells are lysed in the droplet, the target mRNA is reverse transcribed using target-specific primers, and a molecule-specific and droplet-specific barcode is attached to the cDNA by a two-step DNA barcoding process. After subsequent collection and next generation sequencing, this dual barcoding strategy allows sequence reads to be clustered into both their original molecules and cells. Thus, extensive correction of errors and amplification bias, clonal counting at both mRNA and cellular levels, determination of heavy chain isotype, and importantly, V ( D) It becomes possible to collect the J sequence simultaneously with ultra-high throughput.

  Tumor infiltrating lymphocytes (TILs) are important for the anti-cancer immune response, but are difficult to study because their abundance, phenotype, and function are unpredictable. The present inventors have developed a method for characterizing TIL at a high depth without the need for cell sorting, stimulation, or culture. Emulsion-based single cell barcoding captures native paired B cell and T cell receptor (BCR and TCR) sequences from lymphocytes in millions of input cells. In contrast to previous approaches, the variable region recovered is full length and may be accompanied by additional mRNA and protein targets. Before processing 400,000 unsorted cells from ovarian adenocarcinomas and recovering paired BCRs and TCRs from more than 11,000 TILs, 3 million from healthy human blood The method was validated with B cells and 350,000 B cells from HIV patients. Our results are the deepest sampling of any paired BCR or TCR repertoire, and are the first to demonstrate simultaneous RNA sequencing and protein measurement of single cells in an emulsion. .

(Example 3)
Large-scale recovery initial B cell V _H V _L pairs pairs from healthy blood samples, to develop this technology, 3 million to the pairing capability and throughput were isolated from peripheral blood of healthy volunteers by negative bead concentration Of B cells. The emulsion was divided into six separate fractions, which were processed in parallel and not remixed before sequencing. The emulsion was loaded at 0.2 cells per drop. This resulted in a Poisson expectation that about 90% of the occupied droplets contained single cells, consistent with the emulsion droplet observation (FIG. 5A). After emulsion disruption and additional library processing steps, pair-end 325 + 300 bp sequencing was performed using Illumina MiSeq. To process the sequencing data, droplets and molecular barcodes were used together to collect PCR replication reads from each original mRNA molecule, determine consensus for each mRNA, and assembled from at least two reads. Only the mRNA sequence was maintained. The forward and reverse reads were spliced to produce a full-length product containing the 5'UTR, the complete V (D) J sequence, and a constant region sufficient for isotyping. The rearranged immunoglobulin heavy and light chain sequences were annotated using IMGT High-VQuest and / or IgBLAST.

The resulting data set contains 324,988 droplet barcodes that were associated with at least one heavy ( _VH ) and one light ( _VL ) mRNA and was evaluated by heavy chain cluster analysis. There were 229,869 different _VH clone lines. Since this raw set contains data from multiple cells as well as single cell droplets, by filtering out droplet barcodes associated with non-matching heavy or light chain V (D) J sequences, The data from single cell droplets was enriched (FIG. 5B). It is possible to do this due to the high diversity of the typical immune repertoire, with little or no matching of _VH or _VL mRNA from two random cells. Would. The resulting concentrated data set comprises 259,368 pieces of _V H _{V L} droplet barcode contained clone sequence of 182,745 pieces of _{V H.} This arguably indicates that this is the deepest paired immune repertoire sampling to date. Clonally related cells, because their V _H V _L pairing is should be consistent, by identifying the occurrence of clonal expansion was directly assessed the accuracy of the V _H V _L pairing . In more than one emulsion fraction, 2,604 _VH clones that were reliably observed to be clonally expanded cells were identified. The _VL sequences paired with these _VH clones showed very high fractionation consistency and 96.1% pairing accuracy. Thus, it is given a high reliability to the whole filtered data set of 259,368 pieces of _V H _{V L} pair. The cross-fraction _VH and _VL sequences were always associated with a different droplet and molecular barcode in each fraction and therefore did not show library cross-contamination. As some B cells are known to express multiple light chains, analysis may underestimate pairing accuracy. 259,368 pieces of filtered _V H _{V L} droplet barcode or 75.0% of the _"V H _{V L} pair" contained the IgM and / or IgD (Figure 5C). This was often observed together as expected, given that naive naive B cells are typically of the IgM ⁺ IgD ⁺ phenotype. Less was also found a significant proportion of IgA (18.3%) and _{IgG (6.6%) V H V} L pair. All _VH isotypes paired with either Igκ or Igλ in a ratio of about 3: 2. Clonal expansion in the 182,745 _VH clone series was assessed in two ways: the number of droplet barcodes associated with the clone; and the observation of that clone over the emulsion fraction. The appearance of a clone in multiple droplet barcodes may reflect clonal expansion or may reflect multiple barcode droplets. Taking into account that the initial λ = 1, that is, the dispersion of the barcode into the droplet was Poisson dispersion, multiple barcode droplets were expected in about 37% of the droplets. However, all clones represented by> 8 droplet barcodes are likely to be scalable (Poisson probability in a single droplet is <10-6). In total, 6.0% of the clones were found in more than one fraction, while those with more than 8 droplet barcodes (0.7% overall) 99% were found in more than one fraction. All 100 most frequent clones (30-137 droplet barcodes each, FIG. 5D) were found in at least 5 of the 6 fractions. Thus, the combination of barcode counting and independent fraction analysis allows the detection of rare expanded sequences from the vast background of non-expanding clones. However, it is noteworthy that even the largest abundant clones were present in less than one in 1,000 cells, illustrating the very high diversity of the human peripheral immune repertoire. ing.

Number of captured mRNA of each _VH and _VL chain within the pair as an estimate of the expression level (FIG. 5E). In general, less than 10 heavy chain (average 2.0) and light chain (average 4.0) mRNA is captured per droplet barcode, with a few dozen to hundreds of captured heavy and light chains per cell. A subpopulation of droplet barcodes with strand mRNA was observed almost exclusively from IgG and IgA expressing cells. Interestingly, the degree of _VH and _VL mutations within a pair was determined both within each isotype (eg, _VH vs. _VL for IgG) and between isotypes (eg, IgG vs. IgM). Showed a strong correlation (FIG. 2F). In addition, almost all IgG and IgA pairs showed significant mutations in both _VH and _VL chains, whereas most IgM and IgD pairs showed few _VH or _VL mutations. These results are consistent with a mechanism of B cell activation, a class switch from IgM and IgD to IgG or IgA, an increase in immunoglobulin expression, and a system that affects both the heavy and light chain loci of the cell. It leads to cell hypermutation. V _H chains caused a highly mutation, in addition to the observation that tend to pair with V _L chains which caused highly mutated, the method, many derived from resting human B cell repertoire It is possible to generate full length natively matched BCRs.

(Example 4)
Recovery of known low frequency _{VH VL} pair pairs from HIV long-term non-progressors.
As a further validation of the pairing sensitivity and accuracy of the assay, some rare (<1 cell out of 10,000 cells) samples in which the native _{VH VL} pairing was already known were processed. Peripheral B cells were obtained from HIV non-progressive patients. In recent years, antibodies showing HIV neutralizing activity have been searched for in detail in memory B cells of this patient. Treated 350,000 B cells, to produce a total of 38,620 pieces of the filtered _V H _{V L} pair. Interestingly, this individual showed a higher percentage of IgG than previous healthy samples (FIG. 3A) or a typical healthy peripheral B cell repertoire. _VH sequences from this dataset were compared to all reported broadly neutralizing antibodies (bNAbs) from this individual, including PGT121, and eight similar or identical _VH sequences were found. This indicates that this family of bNAbs is less than 0.03% of circulating B cells. Importantly, all the light chains paired with these heavy chains are of the expected similarly rare bNAb series and, as reported previously, have the same Igλ-V3-21 / J3 The rearrangement and characteristic three-codon insertion demonstrated the high accuracy and sensitivity of our method. Furthermore, in the phylogenetic tree of all known newly generated PGT121-like V _H V _L pairs from this individual (FIG. 3B), the V _H and V _L trees show the pairing occupying mirror image-like positions. Shows very similar topologies to the identified _VH and _VL sequences, which likely reflects a common phylogenetic history. The variant pairs found here fit this rule well. Interestingly, the two published antibodies PGT122 and PGT123 were considered an exception, and none were found to support these two pairs, but instead, PGT122V _H : PGT123V _L And PGT123V _H : PGT122V _L- like pairs were found. This represents a match not confirmed in the original report. Eight to synthesize an entire V (D) DNA encoding the J region of the novel PGT like _V H _{V L} pair, to express antibodies as a complete IgG, were tested for their ability to neutralize multiple HIV quasi strains ( (FIG. 6C). Antibodies are well expressed, all exhibit potent neutralizing activity against the virus, and the usefulness of our approach to rapidly generate natively paired functional antibody variants from relevant biological samples. Proven.

(Example 5)
B-cell and T-cell receptor pairs from tumor-infiltrating lymphocytes paired In response to the validation of emulsion barcoding for high-throughput recovery of paired receptors, immunoreceptors could be transferred directly from tumors. Collected. A resected ovarian adenocarcinoma sample separated with protease was obtained and 400,000 unsorted cells were placed in the emulsion. CD3 / CD19 staining of separate equal amounts of the sample indicated that a significant number of invasive B (about 5%) and T cells (about 20%) were present in the material. The single cell dispersion in the emulsion was similar to purified cells, although some limited clumps were visible, and as expected, given the heterogeneous cell type of the sample. There was wide variation in cell size and shape within the drops (FIG. 7A).

Primers targeting both the constant regions of the T-cell receptor alpha and beta chains were used with previously used BCR primers, and after sequencing and stringent filtering, thousands of thousands were linked to the BCR or TCR product. The droplet barcode was collected. To assess single-cell accuracy, all possible combinations of the four target loci ( _VH , _VL , Vα, Vβ) in the droplet barcode were counted (FIG. 7B). Than one majority (97.9%) also drop bar code with many target strand contains pairing of BCR _V ^H + _{V L} or TCR V.alpha ⁺ V? Is biologically expected The mixed BCR-TCR combination contained only 2.1%. Since the barcode assignment of the products is unbiased with respect to the target strand, this result may enhance the reliability of the obtained 6,056 BCR _VH V _L and 5,217 TCR VαVβ pairs. It is possible. BCR showed that IgG (> 80%) was significantly predominant compared to the other isotypes (FIG. 7C), but all were present (IgE <0.05% only). Kappa and lambda light chains were present at similar ratios in the peripheral blood dataset.

Similar to peripheral blood, a correlation was observed between BCR isotypes and mutation levels of both _VH and _VL chains, with IgG and IgA pairs showing larger _VH and _VL mutations than IgD and IgM. And the general correlation of mutations between _VH and _VL within each isotype (Figure 7D). Interestingly, the IgD, IgM, and IgA pairs showed a very similar mutation distribution between the tumor and the peripheral blood dataset (FIG. 5F). Also, the tumor IgG fraction contained a significant proportion of sequences that were not mutated or were not mutated, which were not observed in peripheral blood. In the case of TCR and BCR containing IgD, a similar number of captured mRNA was observed per drop barcode as in peripheral blood BCR results (FIG. 7E, mostly <10 per drop barcode). ). In sharp contrast, tumor-derived IgM, IgA, and IgG pairs showed a 10- to 100-fold increase in average expression levels, with hundreds or thousands of target mRNAs captured in many of the droplet barcodes . Subsequently, the diversity of the captured TIL-TCR and BCR repertoires was assessed (FIG. 7F). Of the 5,217 total TCR pairs, 2,423 different TCR beta clones were observed. Seven clones were present with a frequency of> 1%, with the top clone representing 16.9% of all droplet barcodes. Of the 6,056 total BCR pairs, 1,518 different heavy chain clones were observed, with 15 clones> 1% frequency but none> 5%. This indicates that diversity is substantially more restricted than the healthy peripheral BCR repertoire (no clones were present at more than 0.06% frequency), but did switch classes and mutated mutations. The abundance and abundance of highly expressed clones in tumor samples indicates that a deep and sensitive sampling procedure is needed to characterize TIL. The method allows for the rapid recovery of multiple TIL immunoreceptor pairs simultaneously from both B and T cells without the need to pre-sort or exogenously activate a defined TIL population.

(Example 6)
Capture of additional phenotypic markers of interest Receptor chain pairing by droplet barcoding potentially allows capture of additional targets in addition to immunoreceptors. To explore this possibility, healthy T cells entering the CD4 ⁺ and CD8 ⁺ populations were separated by magnetic bead enrichment, 20,000 cells of each type were placed in separate emulsions, and TCR alpha and beta chains were added. Experiments were performed with targeted primers and CD4 and CD8 mRNA. After sequencing, 47.0% of the 3,861 droplet barcodes containing TCR Vα and Vβ (“TCR pairs”) from CD4 ⁺ isolated cells were associated with CD4 mRNA, Only 0.3% was associated with CD8 mRNA. In contrast, 50.6% of the 2,235 TCR pairs from CD8 ⁺ isolated cells were linked to CD8 mRNA, but only 0.6% were linked to CD4 mRNA. Was. This indicates, as in previous reports, that the mRNA-based approach to determine cell phenotype is highly specific but has limited sensitivity. In contrast, proteins, such as cell surface receptors, are typically present in much larger numbers (1,000-100,000 per cell) than their encoded mRNA, and may be easier to detect. Is likely to be more directly associated with the cellular phenotype. To measure target protein levels in each cell, a custom oligonucleotide DNA label is conjugated to anti-human CD4 and CD8 antibodies, and the labeled antibody is converted to CD4 ⁺ prior to entering 30,000 cells into the emulsion. And with an unseparated mixture of CD8 ⁺ T cells (FIG. 8A). DNA labels carry not only antibody-specific sequence tags, but also molecular barcodes, and sequences complementary to the amplified droplet barcodes, as well as mRNA implementations, emulsion droplet barcodes, and Enables molecule counting. DNA labeling, and TCR, CD4, and CD8 mRNA were simultaneously targeted. After sequencing and filtering, 3,682 droplet barcodes were reliably identified as TCR VαVβ pairs. Consistent with previous experiments, approximately half (52%) of the TCR pairs could be assigned to CD4 or CD8 status based on mRNA (FIG. 8B). However, more than 95% of droplet barcodes can be assigned to CD4 or CD8 based on protein status, and the average molecular count per droplet is that CD4 / 8 protein (20.5 average) / 8 mRNA (average 1.0). Considering that both mRNA and protein determinations were consistent, the match between the mRNA and protein signals was high (FIG. 8C), 96.0% of the droplets. In some rare cases, both CD4 and CD8 proteins were detected. This was thought to be the result of the droplet containing two or more cells. Emulsion barcoding has enabled, for the first time, direct association of single-cell immune receptors with mRNA and protein markers of interest, all at high throughput. The use of this approach to TIL with an expanded set of immuno-oncologically relevant markers, such as anti-PD-1 and anti-CTLA-4, is warranted.

(Example 7)
Human Samples Blood samples for healthy repertoire validation were collected based on Personal Genome Project approval. PBMC for HIV bNAb experiments were obtained from donor 17, an HIV-1 infected donor in the IAVI protocol G cohort. All human HIV samples are from the National Ethics Committee of the Republic of Rwanda, the Institutional Review Board at Emory University, the Research Ethics Committee of the University of Zambia, the Charing Cross Research Ethics Committee, the UVRI Science and Ethics Committee, the University of New South Wales Research Ethics Committee St. Vincent and East Sydney Area Health Services, Kenyatta National Hospital Ethics Committee, Cape Town University Ethics Committee, Institutional Review Board, Mahidol University Ethics Committee, Walter Reed Army Research Institute (WRAIR) Facility Written informed consent based on a clinical trial protocol approved by the Institutional Review Board and the C コ ー ト te d'Ivoire Committee "National d'Ethique des Sciences de la Vie et de la Santa" (CNESVS) Obtained was collected upon. Cryopreserved, dissociated, excised ovarian adenocarcinoma from a single donor was obtained from Convertant Biologics with written informed consent based on an IRB approved protocol.

(Example 8)
Cell Preparation In a study on 3 million healthy B cells, 50 mL of blood was collected into Vacutainer CPT cell preparation tubes with sodium heparin (BD), centrifuged at 1800 × g for 20 minutes, and the cell preparation buffer (2% Washed twice with fetal bovine serum and 1 × PBS supplemented with 2 mM EDTA), removed platelets using 200 × g spin, and obtained PBMCs in RPMI-1640 medium (Life Technologies) + 20% They were stored frozen at -80 ° C in fetal calf serum + 10% DMSO until needed. Prior to emulsion formation, PBMC were thawed, washed twice with cell preparation buffer, and counted. B cells were isolated using a negative selection based human B cell enrichment kit (Stem Cell Technologies) according to the manufacturer's instructions. Pass the cells through a 20 μm cell strainer and use cell preparation buffer at 6.2 × 10 ⁶ cells / mL (experiment with 3 million B cells) or 3.1 × 10 ⁶ cells / mL (with PGT donor and ovarian tumors). Experiment).

(Example 9)
Emulsion Receptor Barcoding in Emulsion The emulsion generation platform provides a computer controlled flow of two aqueous phases and one continuous fluorophilic oil phase into a fluorophilic coated quartz Dolomite mini 2 reagent chip. To enable, there were three Mitos P pumps (Dolomite Microfluidics), each driven by a single air compressor, with a Mitos flow sensor. One aqueous input channel contains the required density of cells to provide the desired level of cell occupancy per droplet, and the second aqueous channel contains the AbPair ™ reaction buffer and oligo. Lysis and nucleotides containing nucleotides (www.abvitro.com/catalog AV2070-1S and AV2080-1S), 5 units / μL MuMLV-based reverse transcriptase (Thermo Scientific), and 0.1 units / μL Herculase II PCR polymerase. It contained the reaction mixture. Using a 100 μL Hamilton Microliter syringe, 100 μL internal diameter PEEK tube sample loops were loaded with approximately 100 μL of each LR mix in two injections. About 100 μL of a 0.2 mm internal diameter FEP tube loop was loaded with about 110 μL of the cell suspension using a 100 μL Hamilton Gastlight syringe. Emulsions were formed by intensive stream injection of the aqueous phase at the same flow rate onto two reagent chips with oil flowing simultaneously from the two oil channels of the chips. The emulsion exiting the chip outlet channel was dropped into a 0.2 mL PCR strip tube (Eppendorf) placed in a cooling block, after which excess oil was removed from the bottom of the tube with a pipette and 40 μL of overlay solution (25 mM Na -EDTA, pH 8.0) was added and the tubes were transferred to a standard thermocycler for the transcription tagging reaction. During a 45 minute reverse transcription (RT) step, RNA was reverse transcribed at 42 ° C. by template-switch based addition of a universal adapter sequence containing a randomized molecular barcode using target-specific RT primers. After RT, the emulsion was subjected to 40 thermal cycles (each cycle: 10 seconds at 82 ° C., 25 seconds at 65 ° C.) to perform PCR amplification of the droplet barcode template, which was performed in the initial lysis and reaction mixture. At 30,000 cp / μL, producing a concentration of 15,000 cp / μL or about 1 final mixture per about 65 pl droplet. One end of the droplet barcode contains an Illumina read 2 ("P7") primer site and the other end matches the consensus sequence of the universal adapter oligonucleotide. Thus, during PCR, the template-switched cDNA anneals to the amplified droplet barcode strand and is spliced by overlap extension to produce a full-length product containing the target, molecular barcode, and droplet barcode sequences. can do.

(Example 10)
Emulsion disruption, cleanup, downstream PCR, pooling, and sequencing After thermal cycling, the overlay solution was pipetted off and 40 μL emulsion disruption solution (1: 1 FC-40: perfluorooctanol) was cleared with 15 μL lysate clearing. Solution (12.5 μL Qiagen protease, 2.5 μL 0.5 M Na-EDTA, pH 8.0). After breaking the emulsion by 10 inversions, the mixture was incubated at 50 ° C. for 15 minutes and at 95 ° C. for 3 minutes to inactivate the protease. After isolating the aqueous phase by centrifugation at 15,000 × g for 1 minute, the recovered material was thoroughly purified to remove oligonucleotides, reagents, and excess droplet barcode PCR products. By cleaning up the streptavidin beads, the full-length products can be efficiently separated from excess droplet barcode PCR products because they contain biotin due to 5 'biotinylation of the RT primer, In addition, downstream PCR recombination artifacts, a common problem with the overlap extension method, are minimized. First, use 1: 1 ratio AMPure XP beads (Agencourt) using the manufacturer's instructions, and then again use streptavidin beads (New England Biolabs) again using the manufacturer's instructions. The product was purified by subsequent elution with deionized water at 95 ° C. followed by a second cleanup with 1: 1 ratio of AMPure XP beads. The product was then placed in target enrichment PCR and primers specific for the constant region of the B or T cell receptor target were used, along with primers specific for the universal end of the droplet barcode sequence. This reverse primer also contained a 6 base index barcode for multiplex sequencing on a MiSeq instrument according to the manufacturer's instructions. Thus, in this step, only the full-length droplet barcoded target sequence is amplified. First, all targets were placed at 98 ° C. for 10 seconds using Q5 hot start polymerase (New England Biolabs) under the manufacturer's recommended conditions, including a 98 ° C. polymerase activation step of 2 minutes at the start of the reaction; Amplified together for 7 cycles at 20 ° C for 20 seconds; 15 seconds at 72 ° C. Thereafter, AMPure XP cleanup was performed at a bead: PCR ratio of 1.5: 1. A second 7 cycles were then performed, targeting each strand ( _VH , _VL , Vα, Vβ) separately, using the same thermal cycling conditions as before, followed by AMPure XP cleanup. went. Then, under the same thermal cycling conditions and 5-15 cycles (depending on the yield determined by qPCR), a full-length Illumina sequencing adapter was added to generate a final PCR that produced enough material for TapeStation D1000 (Agilent) quantification. Carried out. The libraries were then pooled and sequenced on a V3 2x300 bp MiSeq platform (Illumina).

(Example 11)
Modification of the MiSeq Platform Reconstruction of the complete variable V (D) J region of the BCR or TCR requires joining two paired-end Illumina reads. To improve this process, the forward reading of the 2x300 bp kit was extended to 325 bp. The immunoreceptor library used a 10% phiX spike-in to limit the diversity of the constant region primer sites, alleviating the problem of limiting library diversity.

(Example 12)
Overview of Bioinformatics Processing of Reads Using a custom pipeline assembled around the pRESTO package (version 0.4), process Illumina MiSeq reads to generate a full-length consensus sequence of mRNA molecules from each droplet, Annotated with IgBLAST and / or IMGT / HighV-QUEST and further aligned, filtered and processed using custom scripts and the Change-O package to generate statistics.

(Example 13)
Read processing and annotation, isotype assignment.
Raw read processing, V (D) J annotation, and clone assignment were performed in a custom-built pipeline utilizing the pRESTO and Change-O packages. Briefly, raw Illumina paired-end 325 + 300 bp reads were quality controlled, primer trimmed, and droplet-specific (DB) and molecule-specific barcodes (MB) were identified by fuzzy matching of primer sites. In summary, the origin molecules are uniquely identified by DB and MB, and this unique molecular identifier (UMI) is used to group matching PCR replication reads (minimum two) originating from the same molecule, Generate consensus. Isotype-specific priming was confirmed by fuzzy matching of known isotype-specific constant regions within the primer trimmed sequence. V (D) J germline segments and rearranged structures were determined using IgBLAST, confirmed by IMGT / HighV-QUEST and, where appropriate, analyzed by Change-O and custom scripts.

  Clones were assigned by single-chain clustering within the groups of matching IGHV genes, IGHJ genes, and functional V (D) J sequences with junction length, as implemented in Change-O. Was. For the 3 million circulating cell dataset, a 4.0 weighted intra-clone distance was used as the nearest-neighbor distance cut-off using a symmetrized transition / transversion model.

(Example 14)
Droplet immunoreceptor-containing filtering and pairing fidelity calculation The accuracy of recovering B cell sequences from droplets can be determined in two ways with this barcode application: mRNA sequence matching within droplets and cross-fraction pairing matching Assessment. Within each droplet, multiple mRNAs are captured per locus, and the expressed V (D) J sequences from one cell should match. Using a cut-off of 2% sequence diversity (average pairwise nucleotide difference pi55 <0.02) of multiple aligned V (D) J segments defining sequence identity, 1 per droplet The presence of more than one productive VDJ and one productive VJ sequence is bioinformatically marked as putative immunoreceptor containing or multiple cell occupancy. Heavy and light chain consensus sequences were assembled for each allele-excluded droplet and used for clonal definition and cross-fraction pairing analysis. In the 3 million circulating B-cell dataset, each _VH series is associated with one of the> 20,000 light chain clones in the dataset (ideal case). 259,368 pieces of immune loci exclusionary in droplets having a V _H V _L pair, 10,870 pieces of VDJ heavy chain locus rearrangement cluster, at least six physically separated emulsion fractions There were two. These clusters represent either proliferative lineages or independent but similar rearrangements of the same VJ exon. When the VDJ rearrangement was paired with a consistent VJ rearrangement in two replicates, both experiments independently yielded true positives (of 2,604 clones with the rarer rearrangement). 33,157 of 35,922 possible pairwise comparisons). Therefore, the accuracy of each copy is 96.1% (0.923 ＾ 0.5).

(Example 15)
The 38,620 pieces of _V H _{V L} pairs findings the inventors of HIV BNAb candidate sequences, tblastx, MUSCLE, and known BNAb HCDR3 that using PhyML were selected from the literature, VDJ sequence, and the donor 17 series and By searching for similarities of the novel broad-neutralizing antibodies (BNAbs) against the new native of HIV, and then manually examining the phylogenetic tree of the complete V (D) J amino acid sequence, Antibody candidates dispersed with known bNAb sequences were selected.

(Example 16)
HIV bNAb protein expression and purification Antibody sequences were synthesized and cloned into the heavy and light chain vectors previously described. Heavy and light chain plasmids were co-transfected into 293FreeStyle cells (1: 1 ratio) using 293fectin (Invitrogen) according to the manufacturer's protocol. Antibody supernatant was collected 4 days after transfection and purified by protein A affinity chromatography. Purified antibodies were buffer exchanged with PBS before use in further assays.

(Example 17)
Mock virus production and neutralization assay Mock virus was generated by transfecting 293T cells with the HIV-1 Env expression plasmid and the Env deficient genomic backbone plasmid (pSG3ΔEnv). 72 hours after transfection, mock virus was harvested for use in neutralization assays. Neutralizing activity was assessed using a single round of replication mimic virus assay and TZM-B1 target cells. Briefly, TZM-B1 cells were seeded in 96-well flat bottom plates. To this plate, the mock virus was added, which was preincubated for 1 hour at 37 ° C. with serial dilutions of the antibody. The cells were lysed 72 hours after infection and Bright-Glo luciferase substrate (Promega) was added to quantify luciferase reporter gene expression. To determine IC ₅₀ values, the dose response curves were fitted by non-linear regression.

(Example 18)
Identification and identification of ovarian tumor target strands After simultaneous capture of BCR and TCR from ovarian dissociated tumor tissue in the emulsion, the readings were filtered using molecular and droplet barcodes as described previously, but The presence of each of the two possible target strand types (BCR _VH , BCR V _L , TCR Vα, TCR Vβ) was sought. The target strand was retained when supported by at least two mRNAs each having at least two sequencing reads. All droplet barcodes containing only BCR _{VH VL} or TCR VαVβ pairs were further analyzed. BCR heavy chain and TCR beta chain clones were called based on individual CDR3 amino acid sequences.

(Example 19)
Detection of Protein in Emulsion by DNA-Labeled Antibody Staining A single-stranded 200 bp DNA oligonucleotide was designed to contain a unique 5 bp antigen ID sequence and modified with a 5 ′ amino group (Integrated DNA Technologies). Mouse monoclonal anti-human CD4 (BioLegend, # 300516) and CD8a (BioLegend, # 301018) antibodies were conjugated to DNA oligonucleotide tags using a Thunder-Link kit (Innova Biosciences) according to the manufacturer's protocol. For pre-emulsion cell labeling, 2 million negatively selected T cells from peripheral blood were diluted in 400 μL cell buffer + 2 mM EDTA + 0.05% sodium azide. Single-stranded salmon sperm DNA was added to the cells at a final concentration of 200 μg / mL, and the cells were spun at room temperature for 5 minutes. A mixture of CD4 and CD8a DNA labeled antibodies (5 nM each, final concentration) was added to the cells and incubated for 30 minutes at room temperature. Cells were washed three times with cell buffer + 2 mM EDTA + 0.05% sodium azide + 200 μg / mL single-stranded salmon sperm DNA. Prior to entering the emulsion analysis, the cells were resuspended in cell buffer + 0.05% sodium azide. 30,000 cells were used for emulsion sequencing.
(Example 20)
Tetramer-oligonucleotide conjugate staining

  A tetramer-oligonucleotide composed of four MHC-peptide complexes, each having a streptavidin core and a fluorophore was generated. Each tetramer oligonucleotide further included a molecular barcode and / or tetramer ID code for use in quantification as described below. An exemplary fluorescently labeled tetramer-oligonucleotide conjugate is shown in FIG.

Exemplary methods for determining the affinity of two different TCRs are described herein. Two populations of T cells with different known affinities for MHC-peptides are incubated with various concentrations of tetramer-oligonucleotide conjugates, including MHC-peptide tetramers (pMHC). Unbound tetramer-oligonucleotides are washed away, and cells are entered into an emulsion assay and sequenced. The binding signal is calculated for each T cell based on the sequence reading of the oligonucleotide of the tetramer-oligonucleotide conjugate at each concentration of the tetramer-oligonucleotide conjugate (FIG. 19). T cells incubated with higher concentrations of tetramer-oligonucleotide conjugate are expected to show higher binding signals. T cells with a TCR that has a higher affinity for the MHC-peptide of the tetramer-oligonucleotide conjugate are within the effective range of the assay at the same concentration of tetramer-oligonucleotide conjugate. It is expected that the nucleotide conjugate will show a higher binding signal as compared to T cells having a TCR with a lower affinity for the MHC-peptide. For example, in FIG. 19, between the concentrations of log [tetramer] -8 and -4, the binding signal to TCR1 exceeds that of TCR2, and in this assay TCR1 is used for the tetramer reagent used. Indicates that it would be expected to have a higher affinity.
(Example 21)
Tetramer-oligonucleotide conjugate staining compared to conventional MHC-peptide tetramer staining

  Target-specific T cells were generated to provide a positive control for testing the binding specificity of the exemplary tetramer-oligonucleotide conjugate reagents described herein. Exemplary methods for generating target-specific T cells are reviewed in Ho et al., J. Immunol. Methods, 310: 1-2, 40-52. Briefly, HLA-A02 was cultured for two days in the presence of GM-CSF and IL-4 followed by incubation from day 3 in the presence of pro-inflammatory cytokines to produce mature dendritic cells. : Dendritic cells are derived from the adherent fraction of a peripheral blood mononuclear cell (PBMC) sample obtained from a normal human donor with the: 01. On day 4, the resulting mature dendritic cells are harvested, washed and pulsed with the desired target-derived peptide used in MHC-peptide tetramer (pMHC). On day 5, autologous CD8 + T cells from normal human donors are incubated with dendritic cells pulsed with the peptide. On day 8, IFNγ in the culture is measured as an indicator of a culture containing antigen-specific T cells. Cells from reactive co-cultures are selected and restimulated a few times with peptide-pulsed dendritic cells to enrich for antigen-specific T cells. In some embodiments, these antigen-specific T cells are generated from such an enriched population by cell sorting and / or limiting dilution cloning, essentially as described by Ho et al., 2006. Non-target-specific CD8 + T cells were used as a T cell control population.

Target-specific and non-specific CD8 + T cell control populations were incubated with either fluorescently labeled standard MHC-peptide tetramer or various decreasing concentrations of fluorescently labeled tetramer-oligonucleotide conjugate. After incubation, the presence of cells positively stained for a standard MHC-peptide tetramer or tetramer-oligonucleotide conjugate was identified by flow cytometry. Both standard MHC-peptide tetramers and tetramer-oligonucleotide conjugates incubated with non-specific CD8 + T cells showed little or no positive staining (FIG. 21, top row flow cytometry plots). ). Standard MHC-peptide tetramers incubated with target-specific CD8 + T cells showed a strong double positive MHC-peptide tetramer + CD8 + cell population (FIG. 21, bottom row, left flow cytometry plot). The tetramer-oligonucleotide conjugate incubated with target-specific CD8 + T cells shows a strong double-positive tetramer-oligonucleotide conjugate + CD8 + cell population, which is a tetramer-oligonucleotide It decreased with decreasing conjugate concentration (FIG. 21, bottom row, right three flow cytometry plots). This indicates that the tetramer-oligonucleotide conjugate bound to target-specific T cells as well as the standard MHC-peptide tetramer, and that the tetramer-oligonucleotide bound to target-specific T cells It was shown that the nucleotide conjugate was dependent on the concentration of the tetramer-oligonucleotide conjugate.
(Example 22)
Tetramer-oligonucleotide conjugate binding signal

  As outlined in Example 21, target-specific (bound TCR) or non-specific T cells (unbound TCR) were also used to investigate the number of bound tetramer-oligonucleotide conjugates per cell. A variety of MHC-peptide tetramers, including at least one unique oligonucleotide sequence (e.g., a molecular barcode, tetramer ID code, and / or antigen ID code) recognized by the TCRs of the combined TCR population. Unbound and bound TCR cell populations were separately incubated with a concentration of tetramer-oligonucleotide conjugate. Unbound tetramer-oligonucleotide was washed away and cells were entered into an emulsion assay and sequenced. The binding signal was calculated for each tetramer based on the number of sequence reads of the oligonucleotide at each concentration of the tetramer-oligonucleotide conjugate, which is represented graphically in FIG. The results shown in FIG. 21 show that the tetramer-oligonucleotide conjugate is able to specifically recognize and bind to the TCR on primary target T cells in a dose-dependent manner.

Claims (97)

A method for selecting a T cell receptor (TCR),
(A) contacting a plurality of pMHC-oligonucleotide conjugate molecules with a plurality of T cells;
(B) encoding the Bessel bar-encoding polynucleotide or its complement,
(I) an oligonucleotide portion of a pMHC-oligonucleotide conjugate molecule in one of the plurality of vessels, and (ii) a single T cell of one of the plurality of T cells in the vessel. Attaching to the derived TCR polynucleotide;
(C) sequencing said oligonucleotide portion or complement thereof and said TCR polynucleotide or complement thereof, thereby generating sequence information. A method for determining the binding affinity of a T cell receptor (TCR),
(A) contacting a plurality of pMHC-oligonucleotide conjugate molecules with a plurality of T cells;
(B) sequencing the oligonucleotide portion or its complement, thereby generating sequence information;
(C) determining the binding affinity of the TCR to the pMHC oligonucleotide conjugate based on the sequence information. A method for selecting a T cell receptor (TCR),
(A) contacting a plurality of pMHC-oligonucleotide conjugate molecules with a plurality of T cells, wherein the pMHC-oligonucleotide conjugate molecules include a detection moiety;
(B) obtaining an enriched T cell population from the plurality of T cells of (a) based on the detection of the detection portion;
(C) isolating from the enriched population one single T cell bound to a pMHC-oligonucleotide conjugate molecule in a first one of a plurality of vessels;
(D) encoding the Bessel bar-encoding polynucleotide or its complement,
(I) attaching the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule in the first vessel, and (ii) attaching the TCR polynucleotide from the single T cell in the first vessel. When;
(E) sequencing said oligonucleotide portion or complement thereof and said TCR polynucleotide or complement thereof, thereby generating sequence information. A method for selecting a T cell receptor (TCR),
(A) contacting a plurality of pMHC-oligonucleotide conjugate molecules with a plurality of T cells, wherein the pMHC-oligonucleotide conjugate molecules include a detection moiety;
(B) obtaining an enriched T cell population from the plurality of T cells of (a) based on the detection of the detection portion;
(C) isolating from the enriched population one single T cell bound to a pMHC-oligonucleotide conjugate molecule in a first one of a plurality of vessels;
(D) sequencing the oligonucleotide portion or its complement and the TCR polynucleotide or its complement, thereby generating sequence information;
(E) determining the binding affinity of the TCR encoded by the TCR polynucleotide to the pMHC-oligonucleotide conjugate based on the sequence information.   A pMHC-oligonucleotide conjugate wherein the enriched T cell population is higher than the ratio of T cells bound to pMHC-oligonucleotide conjugate molecules in the plurality of T cells to the total T cells after the contacting step. 5. The method of claim 3 or 4, comprising the ratio of T cells bound to the gate molecule to total T cells. A method for selecting a T cell receptor (TCR),
(A) contacting a first plurality of pMHC-oligonucleotide conjugate molecules with a first plurality of T cells at a first pMHC-oligonucleotide conjugate molecule to cell ratio;
(B) providing a second plurality of pMHC-oligonucleotide conjugate molecules with a second plurality of T cells and a second pMHC-oligonucleotide having a lower ratio of said first pMHC-oligonucleotide conjugate molecules to cells. Contacting at a nucleotide conjugate molecule to cell ratio;
(C) the first Vessel bar-encoding polynucleotide or its complement,
(I) an oligonucleotide portion of a pMHC-oligonucleotide conjugate molecule in a first vessel of the plurality of vessels; and (ii) a single T cell of a first T cell population in said first vessel. Attaching to a first TCR polynucleotide from
(D) a second Vessel bar-encoding polynucleotide or its complement,
(I) an oligonucleotide portion of a pMHC-oligonucleotide conjugate molecule in a second vessel of the plurality of vessels; and (ii) a single T of a second population of T cells in the second vessel. Attaching to a second TCR polynucleotide from the cell;
(E) sequencing the oligonucleotide portion or the complement thereof and the first TCR polynucleotide or the complement thereof of the pMHC-oligonucleotide conjugate molecule in the first vessel, whereby the sequence information is obtained. Generating a set of ones;
(F) sequencing the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule or the complement thereof and the second TCR polynucleotide or the complement thereof in the second vessel, whereby the sequence information is obtained. Generating a set of two;
(G) determining a first binding signal of a TCR encoded by the first TCR polynucleotide to the pMHC-oligonucleotide conjugate based on the first set of sequence information; and Determining a second binding signal of a TCR encoded by said second TCR polynucleotide to said pMHC-oligonucleotide conjugate based on said second set of The method, wherein the polynucleotide and said second TCR polynucleotide encode the same TCR.   4. The method of claim 1 or 3, further comprising determining the binding affinity of the TCR encoded by the TCR polynucleotide to the pMHC-oligonucleotide conjugate based on the sequence information.   Determining the binding affinity of the same TCR encoded by the first TCR polynucleotide and the second TCR polynucleotide based on the first binding signal and the second binding signal. The method of claim 6.   7. The method of claim 6, wherein said first and second plurality of pMHC-oligonucleotide conjugate molecules comprise the same peptide.   10. The method of claim 6 or 9, wherein the MHCs of the first and second plurality of pMHC-oligonucleotide conjugate molecules are of the same subtype. A method for selecting a T cell receptor (TCR),
(A) contacting a plurality of pMHC-oligonucleotide conjugate molecules with a plurality of T cells;
(B)
(I) attaching the first vessel bar-encoding polynucleotide or its complement to the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule in the first vessel of the plurality of vessels;
(Ii) attaching a first TCR polynucleotide from a first single T cell of the T cell population in the first vessel;
(Iii) attaching the second vessel bar-encoding polynucleotide or its complement to the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule in the second vessel of the plurality of vessels;
(Iv) attaching a TCR polynucleotide from a second single T cell of the T cell population in the second vessel;
(C)
(I) sequencing the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule or the complement thereof and the first TCR polynucleotide or the complement thereof in the first vessel, whereby the TCR polynucleotide Generating a first set of sequence information comprising a Bessel barcode sequence read and an oligonucleotide Bessel barcode sequence read;
(Ii) sequencing the oligonucleotide portion of the pMHC-oligonucleotide conjugate molecule or the complement thereof and the second TCR polynucleotide or the complement thereof in the second vessel, whereby the TCR polynucleotide vessel Generating a second set of sequence information comprising a barcode sequence read and an oligonucleotide vessel barcode sequence read;
(D) comparing the binding affinity of the TCR encoded by the first TCR polynucleotide to the binding affinity of the TCR encoded by the second TCR polynucleotide, wherein: The binding affinity of the TCR encoded by the TCR polynucleotide of is determined based on the first set of sequence information, and wherein the binding affinity of the TCR encoded by the second TCR polynucleotide is Determined based on the second set of sequence information;
(E) selecting the TCR encoded by the first TCR polynucleotide based on the comparing, wherein the selected TCR encoded by the first TCR polynucleotide comprises: Having a stronger binding affinity than said TCR encoded by the second TCR polynucleotide.   12. The method of claim 11, wherein said first single T cell is from a patient suffering from a disease or condition.   13. The method of claim 12, wherein the second single T cell is from a patient that does not suffer from the disease or condition.   14. The method according to any one of claims 1 or 6 to 13, wherein the pMHC-oligonucleotide conjugate molecule comprises a detection moiety.   15. The method of claim 14, wherein obtaining an enriched T cell population is based on detecting the detection moiety.   16. The method according to claim 14 or claim 15, wherein the detection moiety comprises a fluorophore.   The vessel barcode sequence of the Bessel bar-encoding polynucleotide or its complement in the first vessel is a vessel barcode of the Bessel bar-encoding polynucleotide or amplicon thereof in a second vessel of the plurality of vessels. 17. The method according to any one of the preceding claims, wherein the method differs from the coding sequence.   18. The method of any one of claims 1 to 17, wherein the sequence information comprises a TCR polynucleotide Bessel barcode sequence read and an oligonucleotide Bessel barcode sequence read.   Following the contacting step, a greater percentage of the pMHC-oligonucleotide conjugate molecule bound to the pMHC-oligonucleotide conjugate molecule in the plurality of T cells after the contacting step has bound to the pMHC-oligonucleotide conjugate molecule. 19. The method according to any one of claims 1 or 6 to 18, further comprising obtaining an enriched T cell population comprising T cells.   20. The method of any one of claims 1 to 19, further comprising determining the number of pMHC-oligonucleotide conjugate molecules bound to the single cell.   The oligonucleotide portion of the pMHC-oligonucleotide conjugate comprises a tetrameric molecular barcode (TMB) sequence that is unique to a single pMHC-oligonucleotide conjugate molecule of the plurality of pMHC-oligonucleotide conjugate molecules. 21. A method according to any one of the preceding claims.   22. The method of claim 21, wherein the sequence information comprises a tetramer molecular barcode sequence read.   23. The method of claim 21 or 22, wherein the number of pMHC-oligonucleotide conjugate molecules bound to the single T cell is determined from the sequence information.   24. The method of any one of claims 21 to 23, wherein the number of pMHC-oligonucleotide conjugate molecules bound to the single T cell is determined from the tetramer molecule barcode sequence reading.   25. The method of any one of claims 21 to 24, wherein each pMHC-oligonucleotide conjugate molecule of the plurality of pMHC-oligonucleotide conjugate molecules comprises a unique TMB sequence.   26. The method of any one of claims 1 to 25, wherein the pMHC portion of the pMHC-oligonucleotide conjugate comprises a multimer of pMHC, optionally a tetramer of pMHC.   27. The method according to any one of the preceding claims, wherein the MHC is in a soluble form.   28. The method of any one of claims 1-27, wherein the peptide of the pMHC portion of the pMHC-oligonucleotide conjugate comprises a synthetic peptide.   29. The method of any one of claims 1-28, wherein the pMHC moiety binds to the single T cell T cell receptor (TCR).   30. The method according to any one of the preceding claims, wherein the oligonucleotide portion comprises a tetramer identification sequence (TID).   31. The method of claim 30, wherein the TID is barcoded on the peptide or the pMHC portion of the pMHC-oligonucleotide conjugate.   32. The method of any one of the preceding claims, wherein the vessel bar-encoding polynucleotide is derived from a template vessel bar-encoding polynucleotide in the vessel.   33. The method of any one of claims 1 to 32, further comprising determining characteristics of the single T cell in the vessel based on the sequence information.   34. The method of claim 33, wherein the sequence information comprises the tetramer identification (TID) sequence or its complement and / or comprises the copy number of a molecule having the TID sequence.   35. The method of any one of claims 1 to 34, further comprising washing the plurality of T cells after the contacting.   36. The method of any one of claims 1 or 6 to 35, further comprising isolating the single T cell in the vessel.   37. The method of claim 36, wherein said single T cell is bound to said pMHC-oligonucleotide conjugate prior to the step of isolating.   38. The method of any one of claims 1 to 37, further comprising lysing the single T cell.   39. The method of claim 38, wherein the lysing step is after the single T cell has been isolated in the vessel.   40. The method of any of the preceding claims, wherein the plurality of T cells are a plurality of unsorted T cells.   The vessel barcoding polynucleotide in a first vessel of the plurality of vessels or the complement of the vessel barcode sequence of the complement thereof comprises a vessel barcoding polynucleotide in a second vessel of the plurality of vessels. 41. The method of any one of claims 1 to 40, wherein said method differs from the Bessel barcode sequence of its complement.   42. Any one of the preceding claims, wherein the Bessel barcode sequence of each Bessel barcoding polynucleotide or its complement in a single one of the plurality of vessels comprises the same Bessel barcode sequence. The method described in.   The Bessel barcode sequence of each Bessel barcoding polynucleotide or its complement in any single vessel of the plurality of vessels may be in any other single vessel of the plurality of vessels. 43. The method of any one of claims 1-42, wherein each of the Vessel barcoding polynucleotides or its complement is unique to the Vessel barcode sequence.   The step of attaching the Bessel bar-encoding polynucleotide to the oligonucleotide moiety and the step of attaching the Bessel bar-encoding polynucleotide to the TCR polynucleotide from the single T cell are performed simultaneously. 44. The method according to any one of claims 1 to 43.   45. The method of any one of claims 1-44, further comprising amplifying the oligonucleotide or its complement.   46. The method of any of the preceding claims, further comprising amplifying the TCR cell polynucleotide or its complement.   47. The method of claim 46, wherein the steps of amplifying the oligonucleotide or its complement and amplifying the TCR polynucleotide or its complement are performed simultaneously.   48. The method of any one of claims 44 to 47, wherein the Bessel barcode sequence of the cellular polynucleotide and the Bessel barcode sequence of the oligonucleotide are the same.   49. The method of any one of claims 1 to 48, further comprising pooling the oligonucleotide, its amplification product, or its complement from two or more vessels of the plurality of vessels.   50. The method of claim 49, further comprising pooling the oligonucleotide, its amplification product or its complement, and the TCR polynucleotide or its complement from two or more vessels of the plurality of vessels. .   51. The method of claim 49 or 50, wherein the pooling step is prior to the sequencing step.   52. The method of any one of the preceding claims, wherein the pMHC-oligonucleotide conjugate comprises a plurality of different pMHC-oligonucleotide conjugates.   53. The method of claim 52, wherein each pMHC-oligonucleotide conjugate of the plurality of pMHC-oligonucleotide conjugates comprises a unique tetramer identification (TID) sequence.   54. The method of any one of claims 1-53, wherein the oligonucleotide comprises a fusion sequence, and wherein the attaching comprises attaching the Bessel bar-encoding polynucleotide to the fusion sequence.   55. The method of any one of claims 1 to 54, wherein the oligonucleotide comprises a primer binding sequence.   56. The method according to any one of the preceding claims, wherein the oligonucleotide comprises a constant sequence.   57. The method of any one of claims 1 to 56, further comprising analyzing the oligonucleotide sequence read Bessel barcode sequence.   58. The method of any one of claims 1 to 57, further comprising analyzing a tetramer identification (TID) sequence of the oligonucleotide sequence read.   59. The method of any one of claims 1 to 58, further comprising analyzing a tetramer molecular barcode (TMB) sequence of the oligonucleotide sequence read.   The analyzing comprises determining the frequency of one or more Bessel barcode sequences, one or more TID sequences, one or more tetramer molecular barcode (TMB) sequences, or a combination thereof. 60. The method of claim 59, comprising:   61. The method of claim 59 or 60, wherein the analyzing comprises comparing.   62. The method of any one of claims 57 to 61, further comprising comparing a tetramer identification (TID) sequence of the oligonucleotide sequence read with a tetramer molecular barcode (TMB) sequence of the oligonucleotide sequence read. the method of.   63. The method of any one of claims 44-62, further comprising sequencing the TCR polynucleotide, its complement, its amplification product, or a combination thereof, thereby producing a TCR polynucleotide sequence readout. .   64. The method of claim 63, further comprising comparing an oligonucleotide sequence read with said TCR polynucleotide sequence read.   65. The method of claim 63 or 64, further comprising comparing the Bessel barcode sequence of the oligonucleotide sequence read with the Bessel barcode sequence of the TCR polynucleotide sequence read.   66. The method of any one of claims 63 to 65, further comprising comparing the TCR polynucleotide sequence reads.   67. The method of any one of claims 63 to 66, further comprising analyzing the Bessel barcode sequence of the TCR polynucleotide sequence read.   68. The method of any one of claims 63 to 67, further comprising analyzing the molecular barcode sequence of the TCR polynucleotide sequence read.   69. The method of any one of claims 63 to 68, further comprising determining a characteristic of a cell based on the analyzing or comparing.   70. The method of any one of claims 57 to 69, further comprising selecting a TCR based on the oligonucleotide sequence reading.   71. The method of any one of claims 63 to 70, further comprising selecting a TCR based on the TCR polynucleotide sequence reading.   The vessel barcoding polynucleotide attached to the oligonucleotide and the vessel barcoding polynucleotide attached to the TCR polynucleotide are derived from the same template vessel barcoding polynucleotide in the vessel. 72. The method according to any one of clauses 1 to 71.   73. The method of any one of claims 1 to 72, wherein the Bessel bar-encoding polynucleotide attached to the oligonucleotide is an amplification product of a template Bessel bar-encoding polynucleotide.   74. The method of claim 72 or 73, wherein the Bessel barcoding polynucleotide attached to the TCR polynucleotide is an amplification product of the template Bessel barcoding polynucleotide.   75. The method according to any one of the preceding claims, wherein the vessel comprises a solid support.   75. The method of any of the preceding claims, wherein the vessel does not include a solid support.   77. The method of any one of the preceding claims, wherein each vessel of the plurality of vessels comprises a single cell.   78. The method according to any one of the preceding claims, wherein the vessel is a well, emulsion or droplet.   79. The method of any one of claims 1-78, wherein the template vessel bar-encoding polynucleotide is not attached to a solid support.   79. The method of any one of the preceding claims, wherein the template vessel bar-encoding polynucleotide is attached to a solid support.   Attaching a molecular barcode sequence of a molecular barcoding polynucleotide of the plurality of molecular barcoding polynucleotides to the TCR polynucleotide, wherein the molecular barcode sequence comprises a single TCR polynucleotide molecule and 81. The method of any one of claims 1 to 80, further comprising the step of being barcoded into the amplification product.   82. The method of any one of claims 1-81, wherein the attaching comprises ligating the Bessel bar-coded polynucleotide or its complement to the oligonucleotide.   83. The method of any one of claims 1 to 82, wherein the attaching comprises attaching the Bessel bar-encoding polynucleotide or its complement to the oligonucleotide by an enzyme.   84. The method of any one of claims 1 to 83, wherein the attaching comprises hybridizing the Bessel bar-encoding polynucleotide or its complement to the oligonucleotide.   85. The method of claim 84, wherein said attaching step further comprises extending said oligonucleotide.   86. The method of any one of claims 1-85, wherein said attaching comprises amplifying a template Bessel bar-encoding polynucleotide.   87. The method of any one of claims 1 to 86, wherein the oligonucleotide is double-stranded.   87. The method according to any one of claims 1 to 86, wherein the oligonucleotide is single-stranded.   The method according to any one of claims 1 to 88, wherein the oligonucleotide is DNA.   The method according to any one of claims 1 to 88, wherein the oligonucleotide is RNA.   91. The method of any one of the preceding claims, wherein the cellular polynucleotide comprises a variable region sequence.   92. The method of any one of the preceding claims, further comprising the step of pairing a native TCR chain sequence containing the variable region sequence.   93. The method of any one of claims 1 to 92, wherein said TCR polynucleotide is DNA.   94. The method of any one of claims 1 to 93, wherein the TCR polynucleotide is an RNA.   95. The method according to claim 94, wherein said RNA is mRNA. A composition comprising a plurality of vessels, wherein one vessel of the plurality of vessels comprises:
(A) a single T cell from a sample comprising a plurality of T cells; and (b) a Vessel bar-coded polynucleotide comprising a Vessel bar code sequence, wherein the Vessel comprises a TCR of the single T cell. A composition further comprising a pMHC-oligonucleotide conjugate that binds to. A composition comprising a plurality of vessels, wherein one vessel of the plurality of vessels comprises:
(A) a single lysed T cell from a sample comprising a plurality of T cells; and (b) a pMHC-oligonucleotide conjugate comprising a pMHC portion that binds to a TCR of said single lysed T cell, A composition wherein the oligonucleotide portion of the pMHC-oligonucleotide conjugate comprises a Bessel barcode sequence, and wherein the TCR polynucleotide from the single lysed T cell comprises the same Bessel barcode sequence.